Genetics Lab Manual

Beirut Arab University
Faculty of Health Sciences

Department of Medical Laboratory Technology
GENETICS AND MOLECULAR BIOLOGY LABORATORY (MELS401)
LABORATORY MANUAL (2018-2019)
Said El Shamieh, MLT| PhD.
Said El Shamieh 1
Table of Contents
Experiment 1: Micro-pipetting Basics
Experiment 2: Modeling DNA Structure
Experiment 3: DNA extraction and quantification
Experiment 4: Size Determination of DNA Fragments
Experiment 5: PCR Amplification of DNA
Experiment 6: Design PCR primers
Experiment 7: Identification of Staphylococcus aureus (MRSA and MSSA) using PCR
Experiment 8: VNTR Human DNA Typing Using PCR
Experiment 9: Principles of DNA Sequencing
Experiment 10: Human Karyotype
Said El Shamieh 2
Experiment 1: Micropipetting Basics
Experiment Objective: The objective of this experiment is to learn how to accurately pipet different
microliter volumes using a micropipette and to practice micropipetting solutions of different viscosities.
Said El Shamieh 3
Background Information
ACCURACY AND PRECISION IN BIOTECHNOLOGY

Accuracy describes how close a measurement is to the true value of a given quantity. Precision describes the
reproducibility of the measurement. Accordingly, measurements can be categorized as follows (summarized
in Figure 1):
• Neither accurate nor precise – measurements do not match accepted value, nor are they reproducible.
• Accurate but not precise – the average of the measurements matches the accepted value, but their values
vary greatly.
• Precise but not accurate – the value of the measurements match one another, but the average deviates from
accepted value.
• Both accurate and precise – the measurements agree with one another and with the accepted value.
Accuracy and precision of measurements ensure that your experiments are both successful and reproducible.
Depending upon the procedure being performed, biotechnology experiments can utilize a variety of volumes
of biological samples and reagents, ranging from several hundreds of liters to very small microliter (μl)
volumes.
Advances in biotechnology have resulted in the development and equipment that make these measurements
both accurate and precise.
Pipeting is a critically important technique in life science experiments to ensure accurate experimental
results. For example, small differences in
primer or template concentration can make
a big difference in the results of PCR
experiments.
To address this concern, scientists use
carefully calibrated micropipettes to
measure the volume of each component.
In most biotechnology experiments,
reagents such as DNA, enzymes, and
buffers are transferred by pipeting into
small microcentrifuge tubes that serve as
reaction vessels. For these types of
reactions, microliter volumes are typically
used. There are 1,000 microliters in 1
milliliter of a solution. To put it in
perspective, a 50 microliter sample is
approximately equal in size to a single
raindrop. A raindrop-sized sample is
relatively large when compared to
experimental samples that often are 10 to
50 microliters in volume.
Said El Shamieh 4
Experiment Overview
EXPERIMENT OBJECTIVE:
The objective of this experiment is to learn how to accurately pipet different microliter volumes using a
micropipette and to practice micropipetting solutions of different viscosities.
LABORATORY SAFETY
1. Gloves and goggles should be worn routinely as good laboratory practice.
2. Exercise extreme caution when working with equipment that is used in conjunction with the heating
and/or melting of reagents.
3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS.
4. Exercise caution when using any electrical equipment in the laboratory.
5. Always wash hands thoroughly with soap and water after handling reagents or biological materials in the
laboratory.
LABORATORY NOTEBOOKS:
Scientists document everything that happens during an experiment, including experimental conditions,
thoughts and observations while conducting the experiment, and, of course, any data collected. Today,
you’ll be documenting your experiment in a laboratory notebook or on a separate worksheet.
Before starting the Experiment:

• Carefully read the introduction and the protocol. Use this information to form a hypothesis for this
experiment.
• Predict the results of your experiment.
During the Experiment:

• Record your observations.
After the Experiment:

• Interpret the results – does your data support or contradict your hypothesis?
• If you repeated this experiment, what would you change? Revise your hypothesis to reflect this change.
Said El Shamieh 5
Activity One:
The metric system is used in micropipetting. The milliliter (ml) and microliter (μl) are two very useful units
of measure in molecular biology. “Milli” means one-thousandth and “Micro” means one-millionth. The
symbol “μ” means micro, the prefix for 1 x 10-6 (expressed in scientific notation) or 0.000001 (expressed in
decimals). One microliter is abbreviated as “μl”. The two ways that this would be expressed is:
1μl = .000001 or 1μl = 1 x 10-6. There are 1,000μl in 1 milliliter, and 1,000ml in one liter.
1. Perform the following conversions:
In decimals In scientific notation
1ml = ____________ liter 1ml = ____________ liter
1 liter = ____________ml 1 liter = ____________ml
1ml = ____________μl 1ml = ____________μl
1μl = ____________ml 1μl = ____________ml
10μl = ____________ml 10μl = ____________ml
20μl = ____________ml 20μl = ____________ml
50μl = ____________ml 50μl = ____________ml
100μl = ____________ml 100μl = ____________ml
2. How many times greater is aml than aμl? ______________
3. How many times greater is a liter than aml? ______________
4. How many times greater is a liter than aμl? ______________
Said El Shamieh 6
Activity Two - Option A: Micropipetting Using a Variable Micropipet
To measure microliter volumes, a special instrument called a micropipet is used. The variable automatic
micropipette is the preferred instrument for delivering accurate, reproducible volumes of sample. These
instruments are manufactured to deliver samples in various ranges (e.g., 0.5-10μl, 5-50μl, 200-1000μl, etc.)
and usually can be adjusted in one-microliter increments. Typically, these instruments have an ejector button
for releasing the tip after sample delivery. Variable automatic micropipets can also be multi-channeled,
designed to uniformly deliver several samples at the sample time. However, for this experiment, only one
sample will be delivered at a time.
1. SET the micropipet to the appropriate

volume by adjusting the dial.
2. PLACE a clean tip on the micropipet.
3. PRESS the plunger down to the first stop.
HOLD the plunger down while placing the tip
beneath the surface of the liquid.
4. Slowly RELEASE the plunger to draw
sample into the pipette tip.
5. DELIVER the sample by slowly pressing
the plunger to the first stop. Depress the
plunger to the second stop to expel any
remaining sample.
DO NOT RELEASE the plunger until the tip
is out of the sample container.
6. DISCARD the tip by pressing the ejector
button.
Use a new tip for the next sample.
Said El Shamieh 7
In the activity which follows, you will use a variable micropipet to prepare (mix) seven different dye
mixtures in the wells of a microtiter plate. Each dye mixture will be prepared in triplicate. You will then
pipet 10μl from each well of the microtiter plate onto the Pipet Card.
1. Place the microtiter plate as shown in the figure below, and carefully mark the plate with your initials or
lab group number.
2. Using a permanent marker, label the rows 1 – 7 down the side of the plate.
3. Refer to Table A below to prepare seven dye mixtures, with each dye mixture prepared in triplicate wells
of the microtiter plate.
4. After preparing the seven dye mixtures, pipet 10μl in triplicate from each well of the microtiter plate onto
the appropriate circles on the Pipet Card™. Pipet the dye mixture in the center of each circle in the
appropriate row.
Said El Shamieh 8
Activity Two - Option B: Using a Fixed Volume Micropipet
Accurate pipeting can be achieved using fixed volume micropipets. These types of micropipets are pre-set to
a specific volume. Although the volume of each individual micropipet cannot be changed, fixed volume
micropipets operate similarly to the variable automatic micropipets. Most fixed volume pipets do not have
ejector buttons, so the tips must be removed manually.
In the following activity, you will use one or more fixed volume micropipette to prepare (mix) seven
different dye mixtures in the wells of a microtiter plate. Each dye mixture will be prepared in triplicate. You
will then pipet 10μl from each well of the microtiter plate onto the Pipet card™.
1. Place the microtiter plate as shown in the figure below, and carefully mark the plate with your initials or
lab group number.
2. Using a permanent marker, label the rows 1 – 7 down the side of the plate.
3. Refer to Table B below to prepare seven dye mixtures, with each dye mixture prepared in triplicate wells
of the microtiter plate.
4. After preparing the seven dye mixtures, pipet 10μl in triplicate from each well of the microtiter plate onto
the appropriate circles on the Pipet Card™. Pipet the dye mixture in the center of each circle in the
appropriate row.
Said El Shamieh 9
Experiment 2: Modeling DNA Structure
Experiment Objective: Students investigate the molecular structure of DNA. They analyze historical base-
pairing data and draw conclusions from the data to construct DNA models. They also compare their models
to several images of DNA and discuss what each conveys about the molecular structure of DNA.
Said El Shamieh 10
DNA Structure Deoxyribonucleic acid (DNA) is a large polymer, or macromolecule, made of numerous
repeating monomers, or subunits. These subunits of nucleic acids are called nucleotides. Each nucleotide in
DNA consists of the five-carbon sugar deoxyribose, a phosphate group, and one of four nitrogenous bases.
Because they all contain deoxyribose, the nucleotides in DNA are more specifically called
deoxyribonucleotides. However, most of the time they are simply referred to as nucleotides.
The nitrogenous bases are heterocyclic organic molecules, which means that they are made of ring structures
that include both carbon and another element—in this case, nitrogen. Two of the four nitrogenous bases in
DNA are purines and two are pyrimidines, as shown below.
The complete atomic structure of a nucleotide containing thymine is shown below. The phosphate group is
Said El Shamieh 11
attached to carbon 5 of the deoxyribose sugar, and the bases are attached to carbon 1.
In a DNA molecule, the sugar and phosphate group of successive nucleotides link together to form a sugar–
phosphate backbone, as shown at right. The nitrogenous bases project from this backbone. Each base
hydrogen bonds to only one other nitrogenous base to form a base pair. The purine adenine pairs only with
the pyrimidine thymine via two hydrogen bonds. Similarly, guanine pairs with cytosine via three hydrogen
bonds. These hydrogen bonds between complementary nucleotides link two DNA strands together, making
the resulting DN A molecule double-stranded. This double-stranded molecule takes on a helical shape called
a double helix. It resembles a ladder, with the sides formed by the sugar phosphate backbones and the rungs
formed by successive base pairs.
Because the phosphate groups are always on carbon 5 of the deoxyribose sugar, a single strand of DNA has
a polarity. The end that terminates with a phosphate group is called the 5’ end, while the other end is the 3’
Said El Shamieh 12
end. In a double-stranded DNA molecule, the 5’ end of one strand is opposite the 3’ end of the other strand.
Thus, the two strands of DNA in a double helix are said to be antiparallel.
The Work of Erwin Chargaff

In the late 1940s, Erwin Chargaff, an Austrian biochemist working at Columbia University in New York,
was spurred into action after reading the scientific findings of Oswald Avery. Avery was an American
physician renowned for his studies on disease-causing bacteria. Avery’s research supported the hypothesis
that genes are made of DNA. At the time many leading scientists believed cellular proteins served as the
genetic material because they could not figure out how such a simple molecule as DNA, with only four
different nucleotide subunits, could carry genetic information. Chargaff, in an effort to support Avery’s
findings, changed the work focus of his research team and began investigating the biochemical composition
of DNA. Chargaff’s team used two biochemical analysis techniques that were new at the time:
chromatography, to analyze the nucleic acid content of a sample, and ultraviolet spectrophotometry, to
measure the amount of each nitrogenous base in a DNA sample. These techniques allowed the group to
characterize the ratio of DNA nucleotides in various DNA samples. Chargaff published his first papers
describing their work in 1950. The research group further refined its analysis methods to determine the
nitrogenous base composition of DNA from several organisms, including plants, animals, and bacteria.
From the data generated, the team concluded that the amounts of adenine and thymine in a DNA sample
were approximately equal and that the amounts of cytosine and guanine were also approximately equal. In
addition, the data disproved a widely held idea that DNA is the same in all organisms. The data supported
the idea that the DNA of different organisms is composed of unique ratios of nitrogenous bases. The work of
Chargaff and his team provided critical evidence for the discovery of the double-helical structure of DNA.
James Watson and Francis Crick realized that the data Chargaff collected could be the result of pairing
between A and T and between G and C in complementary strands of DNA. Thus, what later became known
as Chargaff’s rules, that A bonds with T and C bonds with G, were an essential piece of evidence in Watson
and Crick’s discovery of the double-helical structure of DNA.
References
Chargaff, E. & Davidson J., eds. (1955). The nucleic acids. New York: Academic Press.
Chargaff, E., Zamenhof, S., & Green, C. (1950). Human desoxypentose nucleic acid: Composition of human
desoxypentose nucleic acid. Nature 165, 756–757.
Chargaff, E., Lipshitz, R., & Green. C. (1952). Composition of the desoxypentose nucleic acids of four
genera of sea-urchin. Journal of Biological Chemistry 195(1):155–60.
Chargaff, E., Lipshitz, R., Green, C., & Hodes, M. E. (1951). The composition of the desoxyribonucleic acid
of salmon sperm. Journal of Biological Chemistry 192, 223–230
Said El Shamieh 13
Materials needed per group
Said El Shamieh 14
Said El Shamieh 15
Said El Shamieh 16
Said El Shamieh 17
Questions:
Said El Shamieh 18
Experiment 3: DNA extraction and quantification
Experiment Objective: In this experiment, students will learn about the physical nature of
DNA by isolating DNA from whole blood and then by quantifying it using a
spectrophotometer.
Said El Shamieh 19
DNA extraction:
Because animal cells lack cell walls and chloroplasts, as compared with plant cells, it is relatively easy for
the cells to be lysed and DNA to be extracted without chloroplast DNA contamination. The following
protocols describe in detail the isolation of animal DNA that is suitable for restriction enzyme digestion,
genomic library construction and Southern blot analysis.
In order to obtain high-molecular-weight DNA, certain precautions must be taken. All glassware, plastic
pipette tips, centrifuge tubes, cell scrapers, solutions and buffers should be autoclaved or filter-sterilized in
order to avoid DNase contamination. Molecular biology-grade or ultrapure chemicals and reagents are
strongly recommended. Gloves should be worn during isolation procedures, and vigorous shaking should be
avoided to prevent DNA from shearing.
Experiment Overview
In this experiment, students will learn about the physical nature of DNA by isolating DNA from onions and
using the common procedure of DNA spooling.
LABORATORY SAFETY
2. Exercise extreme caution when working with equipment that is used in conjunction with the heating
and/or melting of reagents.
laboratory.

experiment.


Said El Shamieh 20
QIAamp DNA Blood Mini Kit
QIAamp DNA Mini and QIAamp DNA Blood Mini Kits provide fast and easy methods for purification of
total DNA for reliable PCR and Southern blotting. Total DNA (e.g., genomic, viral, mitochondrial) can be
purified from whole blood, plasma, serum, buffy coat, bone marrow, other body fluids, lymphocytes,
cultured cells, tissue, and forensic specimens. The simple QIAamp spin and vacuum procedures, which are
ideal for simultaneous processing of multiple samples, yield pure DNA ready for direct amplification in just
20 minutes. The QIAamp spin procedures can be fully automated on the QIAcube® for increased
standardization and ease of use (see page 14). The QIAamp procedure is suitable for use with fresh or frozen
whole blood and blood which has been treated with citrate, heparin, or EDTA. Prior separation of leukocytes
is not necessary. Purification requires no phenol/chloroform extraction or alcohol precipitation, and involves
very little handling. DNA is eluted in Buffer AE or water, ready for direct addition to PCR or other
enzymatic reactions. Alternatively, it can be safely stored a t –30 to –15°C for later use. The purified DNA
is free of protein, nucleases, and other contaminants or inhibitors. DNA purified using QIAamp Kits is up to
50 kb in size, with fragments of approximately 20–30 kb predominating. DNA of this length denatures
completely during thermal cycling and can be amplified with high efficiency
Purification of DNA from forensic and human identity samples

The QIAamp DNA Investigator Kit is recommended for purification of total (genomic and mitochondrial)
DNA from a wide range of forensic and human identity samples, such as casework or crime-scene samples,
dried blood, bone, and sexual assault samples, swabs, and filters. Purification is fast and efficient, and
purified DNA performs well in downstream analyses, such as quantitative PCR and STR analysis, with high
signal-to-noise ratios. The procedure is designed to ensure that there is no sample-to-sample cross-
contamination.
Principle and procedure QIAamp

DNA Mini and QIAamp DNA Blood Mini Kits are designed for rapid purification of an average of 6 μg of
total DNA (e.g., genomic, viral, mitochondrial) from 200μl of whole human blood, and up to 50 μg of DNA
from 200μl of buffy coat, 5 x 106 lymphocytes, or cultured cells that have a normal set of chromosomes. The
procedure is suitable for use with whole blood treated with citrate, heparin, or EDTA;* buffy coat;
lymphocytes; plasma; serum; and body fluids. Samples may be either fresh or frozen. For larger volumes of
whole blood or cultured cells, we recommend using QIAamp DNA Blood Midi or Maxi Kits. The QIAamp
DNA Mini Kit performs all the functions of the QIAamp DNA Blood Mini Kit, and also allows rapid
purification of DNA from solid tissue. On average, up to 30 μg of DNA can be purified from 25 mg of
various human tissues.
Purification on QIAamp Mini spin columns

The QIAamp DNA purification procedure comprises 4 steps and is carried out using QIAamp Mini spin
columns in a standard microcentrifuge, on a vacuum manifold, or fully automated on the QIAcube. The
procedures are designed to ensure that there is no sample-to-sample cross-contamination and allow safe
handling of potentially infectious samples. QIAamp Mini spin columns fit into most standard
microcentrifuge tubes. In the spin protocol, due to the volume of filtrate, 2ml collection tubes (provided) are
required to support the QIAamp Mini spin column during loading and wash steps. For the vacuum protocol,
a vacuum manifold and a vacuum pump capable of producing a vacuum of –800 to –900 mbar are required.
Eluted DNA can be collected in standard 1.5ml microcentrifuge tubes (not provided).
Said El Shamieh 21
Adsorption to the QIAamp membrane
The lysate buffering conditions are adjusted to allow optimal binding of the DNA to the QIAamp membrane
before the sample is loaded onto the QIAamp Mini spin column. DNA is adsorbed onto the QIAamp silica
Said El Shamieh 22
membrane during a brief centrifugation or vacuum step.
Removal of residual contaminants

DNA bound to the QIAamp membrane is washed in 2 centrifugation or vacuum steps. The use of 2 different
wash buffers, Buffer AW1 and Buffer AW2, significantly improves the purity of the eluted DNA. Wash
conditions ensure complete removal of any residual contaminants without affecting DNA binding.
Elution of pure nucleic acids

Purified DNA is eluted from the QIAamp Mini spin column in a concentrated form in either Buffer AE or
water. Elution buffer should be equilibrated to room temperature (15–25oC) before it is applied to the
column. Yields will be increased if the QIAamp Mini spin column is incubated with the elution buffer at
room temperature for 5 minutes before centrifugation.
Preparation of reagents
QIAGEN Protease stock solution (store at 2–8C)

When using the QIAamp DNA Blood Mini Kit (50), pipet 1.2ml protease solvent* into the vial containing
lyophilized QIAGEN Protease, as indicated on the label. When using the QIAamp DNA Blood Mini Kit
(250), pipet 5.5ml protease solvent into the vial containing lyophilized QIAGEN Protease, as indicated on
the label.
Dissolved QIAGEN Protease is stable for up to 12 months when stored at 2–8C.
Buffer AL† (store at room temperature, 15–25C

Mix Buffer AL thoroughly by shaking before use. Buffer AL is stable for 1 year when stored at room
temperature.
Note: Do not add QIAGEN Protease or proteinase K directly to Buffer AL.
Buffer AW1† (store at room temperature, 15–25C

Buffer AW1 is supplied as a concentrate. Before using for the first time, add the appropriate amount of
ethanol (96–100%) as indicated on the bottle.
Buffer AW1 is stable for 1 year when stored closed at room temperature.
Buffer AW2* (store at room temperature, 15–25C

Buffer AW2 is supplied as a concentrate. Before using for the first time, add the appropriate amount of
ethanol (96–100%) to Buffer AW2 concentrate as indicated on the bottle.
Buffer AW2 is stable for 1 year when stored closed at room temperature.
Protocol: DNA Purification from Blood or Body Fluids

(Spin Protocol)
This protocol is for purification of total (genomic, mitochondrial, and viral) DNA from whole blood,
plasma, serum, buffy coat, lymphocytes, and body fluids using a microcentrifuge. For total DNA
purification using a vacuum manifold, see “Protocol:
Important points before starting

■ All centrifugation steps are carried out at room temperature (15–25C).
■ Use carrier DNA if the sample contains <10,000 genome equivalents.
Said El Shamieh 23
■ 200μl of whole blood yields 3–12 μg of DNA. Preparation of buffy coat is recommended if a higher yield
is required.
Things to do before starting
■ Equilibrate samples to room temperature (15–25ÅãC).
■ Heat a water bath or heating block to 56ÅãC for use in step 4.
■ Equilibrate Buffer AE or distilled water to room temperature for elution in step 11.
■ Ensure that Buffer AW1, Buffer AW2, and QIAGEN Protease have been prepared according to the
instructions on page 16.
■ If a precipitate has formed in Buffer AL, dissolve by incubating at 56C.
Procedure
1. Pipet 20μl QIAGEN Protease (or proteinase K) into the bottom of a 1.5ml microcentrifuge tube.
2. Add 200μl sample to the microcentrifuge tube. Use up to 200μl whole blood, plasma, serum, buffy
coat, or body fluids, or up to 5 x 106 lymphocytes in200μl PBS.
If the sample volume is less than 200μl, add the appropriate volume of PBS.
QIAamp Mini spin columns copurify RNA and DNA when both are present in the sample. RNA may inhibit
some downstream enzymatic reactions, but not PCR.
If RNA-free genomic DNA is required, 4μl of an RNase A stock solution (100 mg/ml) should be added to
the sample before addition of Buffer AL.
Note: It is possible to add QIAGEN Protease (or proteinase K) to samples that have already been dispensed
into microcentrifuge tubes. In this case, it is important to ensure proper mixing after adding the enzyme.
3. Add 200μl Buffer AL to the sample. Mix by pulse-vortexing for 15 s.

To ensure efficient lysis, it is essential that the sample and Buffer AL are mixed thoroughly to yield a
homogeneous solution.
If the sample volume is larger than 200μl, increase the amount of QIAGEN
Protease (or proteinase K) and Buffer AL proportionally; for example, a 400μl sample will require 40μl
QIAGEN Protease (or proteinase K) and 400μl Buffer
AL. If sample volumes larger than 400μl are required, use of QIAamp DNA Blood
Midi or Maxi Kits is recommended; these can process up to 2ml or up to 10ml of sample, respectively.
4. Incubate at 56C for 10 min.

DNA yield reaches a maximum after lysis for 10 min at 56C. Longer incubation times have no effect on
yield or quality of the purified DNA.
5. Briefly centrifuge the 1.5ml microcentrifuge tube to remove drops from the inside of the lid.
6. Add 200μl ethanol (96–100%) to the sample, and mix again by pulse-vortexing for 15 s. After
mixing, briefly centrifuge the 1.5ml microcentrifuge tube to remove drops from the inside of the lid.
If the sample volume is greater than 200μl, increase the amount of ethanol proportionally; for example, a
400μl sample will require 400μl of ethanol.
7. Carefully apply the mixture from step 6 to the QIAamp Mini spin column (in a 2ml collection tube)
without wetting the rim. Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the
Said El Shamieh 24
QIAamp Mini spin column in a clean 2ml collection tube (provided), and discard the tube containing
the filtrate.*
Close each spin column to avoid aerosol formation during centrifugation.
Centrifugation is performed at 6000 x g (8000 rpm) to reduce noise. Centrifugation at full speed will not
affect the yield or purity of the DNA. If the lysate has not completely passed through the column after
centrifugation, centrifuge again at higher speed until the QIAamp Mini spin column is empty.
Note: When preparing DNA from buffy coat or lymphocytes, centrifugation at full speed is recommended to
avoid clogging.
8. Carefully open the QIAamp Mini spin column and add 500μl Buffer AW1 without wetting the rim.
Close the cap and centrifuge at 6000 x g (8000 rpm) for 1 min.
Place the QIAamp Mini spin column in a clean 2ml collection tube (provided), and discard the collection
tube containing the filtrate.* It is not necessary to increase the volume of Buffer AW1 if the original sample
volume is larger than 200μl.
Close the cap and centrifuge at full speed (20,000 x g; 14,000 rpm) for 3 min.
10. Recommended: Place the QIAamp Mini spin column in a new 2ml collection tube (not provided)
and discard the old collection tube with the filtrate. Centrifuge at full speed for 1 min.
This step helps to eliminate the chance of possible Buffer AW2 carryover.
11. Place the QIAamp Mini spin column in a clean 1.5ml microcentrifuge tube (not provided), and
discard the collection tube containing the filtrate. Carefully open the QIAamp Mini spin column and
add 200μl Buffer AE or distilled water.
Incubate at room temperature
Said El Shamieh 25
DNA quantification:
Assessment of the quality and concentration of the isolated DNA is essential to all subsequent
manipulations. DNA integrity following extraction, or the efficiency of restriction enzyme digestion, can
only be monitored by agarose-gel electrophoresis. A rough estimate of the concentration of a DNA band of
interest can be obtained on an ethidium bromide (EtBr)-stained gel by loading molecular weight markers of
known DNA concentration in an adjacent lane. This procedure works well for most recombinant DNA
applications where an estimate of DNA concentration is sufficient. One should avoid over-loading DNA, as
the concentration of EtBr-saturated bands is very difficult to estimate by eye. DNA yield, concentration
and/or purity can be determined spectrophotometrically by measuring UV light absorbance (A) of samples
at wavelengths of 260 and 280 nm. Ratios of the absorbencies of these wavelengths at these wavelengths
(A260/A280) provide a gauge of protein contamination in a DNA sample, while the absorbance at 260 nm
(A260/A280) is used to calculate the concentration of DNA in the sample.
UV light absorbance, however, cannot be used to distinguish DNA from contaminating with protein, phenol,
oligo- or polysaccharides. Applications that depend on accurate quantification of very small amounts of
DNA require fluorometry. Several DNA-specific fluorescent dyes are available, and measurement is more
sensitive than with UV absorbance spectrophotometry.
Spectrophotometric measurement
The protocol described below is for general reference. Readers should refer to the instruction manual for
proper operation of their spectrophotometer.
1. Turn on the UV spectrophotometer and set the wavelengths at 260 and 280 nm according to the
instructions. Some newer models of spectrophotometers contain a computerized monitoring unit that can
automatically and simultaneously measure a DNA sample at wavelengths of 260 and 280nm, and calculate
and display the DNA concentration together with the ratio of A260/A280 . If such functions are not available,
program the spectrophotometer to measure and record the A260 and A280 readings for each sample manually.
2. Set up a reference using a blank solution. Depending on the DNA sample, add 0.5ml of the elution buffer
(pH 8.0) or ddH O to a clean 1-ml cuvette or to each of two 1-ml cuvettes (one-half to threequarters full),
depending on whether one or two cells are available in the spectrophotometer. Insert the cuvette(s) into
cuvette holder(s) in the sample compartment with the optical (clear) sides of the cuvette(s) facing the light
path. Close the sample compartment cover and adjust the number 0.000 (either manually or by pressing the
‘Auto Zero’ button, according to manufacturer’s instructions)
3. In a clean microcentrifuge tube, dilute the DNA sample in blank solution to a total of 0.5ml for
measurement. Briefly vortex the sample to mix and transfer the sample to a clean cuvette by simply pouring
or using a disposable Pasteur pipette.
4. Insert the sample cuvette and read the absorbance of the sample cuvette against the blank cuvette
according to the spectrophotometer instructions. If only one cell is available, remove the reference cuvette
after adjusting the absorbance to 0.000 and insert the sample cuvette. Record the reading.
5. After measuring DNA sample(s) at 260 and 280 nm, calculate the ratio A260/A280 and concentration for
each DNA sample. A clean DNA preparation should have an A /A ratio of 1.7 – 2.0. The concentration of
DNA sample is calculates as follows:
Said El Shamieh 26
Experiment 4: Size Determination of DNA Fragments
Experiment Objective: The objective of this experiment module is to develop an understanding of
principles involved in estimating the size of unknown DNA fragments by agarose gel electrophoresis.
Said El Shamieh 27
Size determination of DNA fragments is essential to DNA mapping and analyzing restriction enzyme
cleavage patterns. Restriction enzymes are endonucleases that cleave both strands of DNA at very specific
sequences within DNA. Locations of their cleavage sites are important for DNA fingerprinting,
determination of genetic diseases and for DNA analysis.
Agarose gel electrophoresis is a convenient analytical method for determining the size of DNA molecules in
the range of 500 to 30,000 base pairs. Samples of DNA are delivered in wells made in an agarose gel, which
is placed in an electrophoresis chamber containing a buffer solution and electrodes. Direct current (D.C.) is
applied from a power source. Since DNA is negatively charged at neutral pH, it will migrate through the gel
towards the positive electrode. The agarose gel consists of microscopic pores that act as a molecular sieve
that separates DNA molecules according to their size and shape. The migration rate of DNA molecules of
the same shape is inversely proportional to their size. This results in smaller DNA molecules to migrate
faster through the gel. The charge to mass ratio is the same for different sized DNA molecules.
Nucleotides in DNA are linked together by negatively charged phosphodiester bonds. For every base pair
(average molecular weight of approximately 660) there are two charged phosphodiester linkages. Therefore,
negative charges in DNA is accompanied by approximately the same mass. The absolute amount of charge
in DNA is not a critical factor in the separation process. Separation occurs because smaller molecules pass
through the gel pores more easily than larger ones (i.e., the gel is sensitive to the physical size of the
molecule). DNA fragment migration rate is inversely proportional to the log10 of its size in base pairs.
In this experiment, DNA fragments of unknown size and Standard DNA fragments are submitted to
electrophoretic separation. The unknown DNA fragments will migrate through the gel according to their
respective sizes and relative to the Standard DNA fragments. After electrophoresis, the gel is stained and the
DNA bands are visualized. The migration distances of the known and unknown fragments are measured and
plotted on semi-log graph paper according to their size on the y-axis versus the migration distance on the x-
axis. The size of the fragments on the y-axis are expressed as the log of the number of base pairs. This
allows the data to be plotted as a straight line. The DNA fragments of known size (Standard DNA
fragments) are used to plot a standard curve. The migration distance of the unknown DNA fragments are
estimated by extrapolation from the standard curve.
The standard fragments are used to make a standard curve by plotting their size on the y-axis versus the
migration distance on the x-axis. The size of the fragments on the y-axis are expressed as the log of the
number of base pairs they contain or the log of their molecular weight. Most of the plotted data obtained
from the markers will yield a straight line. The migration distance of the unknown DNA fragment(s) are
found on the X-axis and their size is estimated from the standard curve.
After determining the size of the DNA fragments generated by single and combinations of restriction
enzymes, a DNA map is constructed as previously described.
In this experiment, you will determine the relative locations of three restriction enzyme cleavage sites on a
circular plasmid DNA. The plasmid has been cleaved with two restriction enzymes. Enzyme 1 cleaves the
plasmid once. Assume that the Enzyme 1 site is at position 0. Enzyme 2 cuts the plasmid twice. The
objective is to calculate the distances in base pairs between the points of cleavage and to determine whether
the Enzyme 1 site is in between the Enzyme 2 sites.
Said El Shamieh 28
Experiment Overview
Said El Shamieh 29
The objective of this experiment module is to develop an understanding of principles involved in estimating
the size of unknown DNA fragments by agarose gel electrophoresis.
LABORATORY SAFETY
2. Exercise extreme caution when working with equipment that is used in
conjunction with the heating and/or melting of reagents.
laboratory.

experiment.


Said El Shamieh 30
Said El Shamieh 31
CASTING THE AGAROSE GEL
1. DILUTE concentrated 50X Electrophoresis buffer with distilled water (refer to Table A for correct
volumes depending on the size of your gel casting tray).
2. MIX agarose powder with buffer solution in a 250ml flask (refer to Table A).
3. DISSOLVE agarose powder by

boiling the solution.
MICROWAVE the solution on
high for 1 minute. Carefully
REMOVE the flask from the
microwave and MIX by swirling
the flask. Continue to HEAT the
solution in 15-second bursts until
the agarose is completely dissolved
(the solution should be clear like
water).
4. COOL agarose to 60C with careful swirling to promote even dissipation of heat.
5. While agarose is cooling, add 4 uL of Ethidium bromide dye.
Said El Shamieh 32
6. POUR the cooled agarose solution into the prepared gel-casting tray. The gel should thoroughly solidify
within 20 minutes. The gel will stiffen and become less transparent as it solidifies.
7. REMOVE end caps and comb. Take particular care when removing the comb to prevent damage to the
wells.
RUNNING THE GEL

8. PLACE the gel (still on the tray) into the electrophoresis chamber. COVER the gel with 1X
Electrophoresis Buffer. The gel should be completely submerged.
9. PLACE safety cover on the unit. CHECK that the gel is properly oriented. Remember, the DNA samples
will migrate toward the positive (red) electrode.
10. CONNECT leads to the power source and PERFORM electrophoresis (See Table C for time and voltage
guidelines). Allow the tracking dye to migrate at least 3.5 cm from the wells.
11. After electrophoresis is complete, REMOVE the gel and casting tray from the electrophoresis chamber
and proceed to instructions for revelation on UV lamp.
12. TRANSFER the gel to the UV Lamp.
13. VISUALIZE results using a white light visualization system. DNA will appear as purple bands on a dark
background
Said El Shamieh 33
Module III: Size Determination of DNA Fragments
Agarose gel electrophoresis separates biomolecules into discrete bands, each comprising molecules of the
same size. How can these results be used to determine the lengths of different fragments? Remember, as the
length of a biomolecule increases, the distance to which the molecule can migrate decreases because large
molecules cannot pass through the channels in the gel with ease. Therefore, the migration rate is inversely
proportional to the length of the molecules—more specifically, to the log10 of molecule's length. To
illustrate this, we ran a sample that contains bands of known lengths called a “standard”. We will measure
the distance that each of these bands traveled to create a graph, known as a “standard curve”, which can then
be used to extrapolate the size of unknown molecule(s).
1. Measure and Record Migration Distances

Measure the distance traveled by each
Standard
DNA Fragment from the lower edge of
the sample well to the lower end of
each band. Record the distance in
centimeters (to the nearest millimeter)
in your notebook.
Repeat this for each DNA fragment in
the standard.
Measure and record the migration
distances of each of the fragments in
the unknown samples in the same way
you measured the standard bands.
2. Generate a Standard Curve.
Because migration rate is inversely
proportional to the log10 of band
length, plotting the data as a semi-log
plot will produce a straight line and
allow us to analyze an exponential
range of fragment sizes. You will
notice that the vertical axis of the
semilog plot appears atypical at first;
the distance between numbers shrinks
as the axis progresses from 1 to 9. This
is because the axis represents a
logarithmic scale. The first cycle on
the y-axis corresponds to lengths from
100-1,000 base pairs, the second cycle
measures 1,000-10,000 base pairs, and
so on. To create a standard curve on
the semilog paper, plot the distance
each Standard DNA fragment migrated
on the x-axis (in mm) versus its size
on the y-axis (in base pairs). Be sure to
label the axes!
Said El Shamieh 34
After all the points have been plotted, use a ruler or a straight edge to draw the best straight line possible
through the points. The line should have approximately equal numbers of points scattered on each side of
the line. It is okay if the line runs through some points (see Figure 1 for an example).
3. Determine the length of each unknown fragment.

a. Locate the migration distance of the unknown fragment on the x-axis of your semi-log graph. Draw a
vertical line extending from that point until it intersects the line of your standard curve.
b. From the point of intersection, draw a second line, this time horizontally, toward the y-axis. The value at
which this line intersects the y-axis represents the approximate size of the fragment in base pairs (refer to
Figure 1 for an example). Make note of this in your lab notebook.
c. Repeat for each fragment in your unknown sample.
Said El Shamieh 35
Said El Shamieh 36
Study Questions
1. How should the x and y axes of the semi-log graph paper used in this experiment, be labeled?
2. Determine Unknown 1 and Unknown 2 DNA fragment sizes in base pairs according to your standard
curve, then determine your percentage error.
3. Use the example of the standard curve to determine:
a. The base pair size if the migration distance is 2.8 centimeters.
b. The migration distance if the fragment contains 5,500 base pairs.
Said El Shamieh 37
Experiment 5: PCR Amplification of DNA
Experiment Objective: The objective of this experiment is for students to gain hands-on experience of the
principles and practice of Polymerase Chain Reaction (PCR). The PCR products are then analyzed by
agarose gel electrophoresis.
Said El Shamieh 38
THEORY OF PCR
The polymerase chain reaction (PCR) is a DNA amplification technique that has revolutionized almost all
aspects of biological research. PCR was invented in 1984 by Dr. Kary Mullis at the Cetus Corporation in
California. The enormous utility of the PCR method is based on its ease of use and its ability to allow the
amplification of small DNA fragments. For this ground breaking technology, Mullis was awarded the Nobel
Prize in Chemistry in 1993.
Before performing PCR, template DNA is extracted from various biological sources. Because PCR is very
sensitive, only a few copies of the gene are required. Nevertheless, freshly isolated DNA will provide better
amplification results than older DNA specimens that may have become degraded. In order to amplify the
specific DNA or target sequence, two primers (short, synthetic DNA molecules) are designed to correspond
to the ends of the target sequence. The primers hybridize to the DNA template, which marks this sequence
to be copied by DNA Polymerase. Starting from the primer, DNA Polymerase builds a new strand of DNA
in the 5' -> 3' direction, using the DNA template as a guide.
To perform PCR, the template DNA and a molar excess of primers are mixed with the four “free"
deoxynucleotides (dATP, dCTP, dGTP, and dTTP), and a thermostable DNA polymerase. The most
commonly used DNA polymerase is Taq DNA polymerase. This enzyme, originally purified from a
bacterium that inhabits hot springs, is stable at very high temperatures. These components (template DNA,
primers, the four deoxynucleotides, and Taq DNA polymerase) are mixed with a buffer that contains Mg+2,
an essential cofactor for Taq polymerase. The PCR reaction mixture is subjected to sequential
heating/cooling cycles at three different temperatures in a thermal cycler.
• In the first step, known as “denaturation", the mixture is heated to near boiling (94C - 96C) to “un-zip" (or
melt) the target DNA. The high temperature disrupts the hydrogen bonds between the two complementary
DNA strands and causes their separation.
• In the second step, known as “annealing", the reaction mixture is cooled to 45C - 65C, which allows the
primers to base pair with the target DNA sequence.
• In the third step, known as “extension", the temperature is raised to 72C. This is the optimal temperature at
which Taq polymerase can add nucleotides to the hybridized primers to synthesize the new complementary
strands.
These three steps - denaturation, annealing, and extension - constitute one PCR “cycle" (Figure 1). Each
PCR cycle doubles the amount of the target DNA in less than five minutes. In order to produce enough
DNA for analysis, twenty to forty cycles may be required. To simplify this process, a specialized machine,
called a “thermal cycler" or a “PCR machine", was created to rapidly heat and cool the samples.
PRACTICE OF PCR Mathematically, PCR can be expressed as an exponential relationship – if we begin
with a starting copy number of m, then after n cycles, we will have m x 2n copies of our DNA target. For
example, in Figure 1 we start with one copy of our template DNA. After three rounds of PCR, we end up
with eight copies of the DNA template. If the amplification continued for a total of 20 cycles, the
polymerase chain reaction would have produced over one million copies of the original DNA template. In
theory, this process could continue indefinitely. In practice, after many cycles (regardless of the amount of
DNA present in the starting material) the amount of DNA produced reaches a maximum where a product
curve flattens out, known as the plateau (Figure 2). This leveling off of the curve is due to the depletion of
reaction components like primers and nucleotides and the loss of Taq polymerase activity.
The exact temperature and incubation time required for each step depends on several factors, including the
length of the target DNA and GC content of the primer/template. In some cases, the annealing and extension
steps may be combined resulting in a two step PCR experiment. One common problem that occurs during
Said El Shamieh 39
PCR is unwanted amplification products. These are due to contamination of the sample or nonspecific
annealing of the primers. To reduce contamination, autoclaved tubes, pipet tips, and sterile water should be
used. Gloves should always be worn when performing PCR. To minimize unwanted PCR products due to
nonspecific primer annealing, the primer concentration should be minimized, if possible. Another common
technique is “hot start", in which the components of the PCR reaction are fully mixed only after the DNA is
fully denatured above 94C.
Because of its ease of use and its ability to rapidly amplify DNA, PCR has become indispensable in medical
and life sciences labs, replacing as the method of choice more time- and labor-intensive methods such as
Southern blotting. For example, today’s research laboratories can quickly create copies of a specific region
of DNA for cloning applications. Medical diagnostics use PCR to identify genetic mutations and infectious
Said El Shamieh 40
agents. In addition, because amplification by PCR requires very little starting material, it is ideal for forensic
analysis of biological samples or determination of paternity.
The objective of this experiment is for students to gain hands-on experience of the principles and practice of
Polymerase Chain Reaction (PCR). The PCR products are then analyzed by agarose gel electrophoresis.
LABORATORY SAFETY:
Be sure to READ and UNDERSTAND the instructions completely BEFORE starting the experiment. If you
are unsure of something, ASK YOUR INSTRUCTOR!
• Wear gloves and goggles while working in the laboratory.
• Exercise caution when working in the laboratory – you will be using equipment that can be dangerous if
used incorrectly.
• Wear protective gloves when working with hot reagents like boiling water and melted agarose.
• Always wash hands thoroughly with soap and water after working in the laboratory.
thoughts and observations while conducting the experiment, and, of course, any data collected. Today, you'll
be documenting your experiment in a laboratory notebook or on a separate worksheet.

experiment.


Said El Shamieh 41
Module I: Performing the Polymerase Chain Reaction
Taq PCR Master Mix Kit
Taq PCR Master Mix provides a final concentration of 1.5mM MgCl2 in the reaction mix, which will give
satisfactory results in most cases.
Recommended PCR reaction mix:
Component Volume/reaction
Reaction mix 10μl
Forward Primer (10μM) 1μl
Reverse Primer (10μM) 1μl
Template DNA 100ng
RNase-free water (provided) up to 20μl
Recommended PCR cycles:
Step Time Temperature

Initial denaturation 3 min 94°C
3-step cycling:
Denaturation 0.5–1 min 94°C
Annealing 0.5–1 min 50–68°C
Extension 1 min 72°C
Number of cycles 25–35
Final extension 10 min 72°C
IMPORTANT: After amplification, samples can be stored overnight at 2–8°C, or at –20°C for longer
storage.
Said El Shamieh 42
Module II: Separation of PCR Products by Electrophoresis
1. DILUTE concentrated (50X) buffer with distilled water to create 1X buffer (see Table A).
2. MIX agarose powder with 1X buffer in a 250ml flask (see Table A).
3. DISSOLVE agarose powder by boiling the solution. MICROWAVE the solution on high for 1 minute.
Carefully REMOVE the flask from the microwave and MIX by swirling the flask. Continue to HEAT the
solution in 15-second bursts until the agarose is completely dissolved (the solution should be clear like
water).
5. While agarose is cooling, add 4uL of Ethidium bromide. PLACE the well template (comb) in the
appropriate notch.
6. POUR the cooled agarose solution into the

prepared gel-casting tray. The gel should thoroughly
solidify within 20 minutes. The gel will stiffen and
become less transparent as it solidifies.
7. REMOVE end caps and comb. Take particular

care when removing the comb to prevent damage to
the wells.
8. PLACE gel (on the tray) into electrophoresis chamber. COVER the gel with 1X electrophoresis buffer
Said El Shamieh 43
(See Table B for recommended volumes). The gel should be completely submerged.
9. LOAD the entire volume (25μl) into the well.
10. PLACE safety cover. CHECK that the gel is properly oriented. Remember, the DNA samples will
migrate toward the positive (red) electrode.
11. CONNECT leads to the power source and PERFORM electrophoresis.
and proceed to STAINING the agarose gel.
background.
Said El Shamieh 44
Module IV: Size Determination of Amplified PCR Products
Agarose gel electrophoresis separates DNA molecules into discrete bands, each comprising molecules of the
same size. How can these results be used to determine the lengths of different fragments? Remember, as the
length of a DNA molecule increases, the distance to which the molecule can migrate decreases because large
molecules cannot pass through the channels in the gel with ease. Therefore, the migration rate is inversely
proportional to the length of the molecules—more specifically, to the log10 of molecule's length. To
illustrate this, we ran a sample that contains bands of known lengths called a “standard". We will measure
the distance that each of these bands traveled to create a graph, known as a “standard curve", which can then
be used to extrapolate the size of unknown molecule(s).
1. Measure and Record Migration

Distances
Measure the distance traveled by each
Standard
DNA Fragment from the lower edge of
the sample well to the lower end of
each band. Record the distance in
centimeters (to the nearest millimeter)
in your notebook.
Repeat this for each DNA fragment in
the standard.
Measure and record the migration
distances of each of the fragments in
the unknown samples in the same way
you measured the standard bands.
2. Generate a Standard Curve.

Because migration rate is inversely
proportional to the log10 of band
length, plotting the data as a semi-log
plot will produce a straight line and
allow us to analyze an exponential
range of fragment sizes. You will
notice that the vertical axis of the
Said El Shamieh 45
semilog plot appears atypical at first; the distance between numbers shrinks as the axis progresses from 1 to
9. This is because the axis represents a logarithmic scale. The first cycle on the y-axis corresponds to lengths
from 100-1,000 base pairs, the second cycle measures 1,000-10,000 base pairs, and so on. To create a
standard curve on the semilog paper, plot the distance each Standard DNA fragment migrated on the x-axis
(in mm) versus its size on the y-axis (in base pairs). Be sure to label the axes!
After all the points have been plotted, use a ruler or a straight edge to draw the best straight line possible
through the points. The line should have approximately equal numbers of points scattered on each side of
the line. It is okay if the line runs through some points (see Figure 1 for an example).
3. Determine the length of each unknown fragment.

a. Locate the migration distance of the unknown fragment on the x-axis of your semi-log graph. Draw a
vertical line extending from that point until it intersects the line of your standard curve.
b. From the point of intersection, draw a second line, this time horizontally, toward the y-axis. The value at
which this line intersects the y-axis represents the approximate size of the fragment in base pairs (refer to
Figure 1 for an example). Make note of this in your lab notebook.
c. Repeat for each fragment in your unknown sample.
Said El Shamieh 46
Said El Shamieh 47
Study Questions
1. Why is a thermostable DNA polymerase required for DNA amplification by PCR?
2. If starting with one copy of the DNA template, how many copies of the DNA template have been
produced after four complete cycles of PCR? After 8 cycles?
3. Why are two different primers required for the PCR reaction?
4. What are possible reasons for obtaining fainter (usually smaller) bands besides the main PCR product?
Said El Shamieh 48
Experiment 6: Design PCR primers
Experiment Objective: In this experiment, students will learn how to pick adequate primers to perform the
PCR amplification process.
Said El Shamieh 49
Said El Shamieh 50
Said El Shamieh 51
Said El Shamieh 52
Said El Shamieh 53
Said El Shamieh 54
Said El Shamieh 55
Said El Shamieh 56
Said El Shamieh 57
Said El Shamieh 58
Said El Shamieh 59
Said El Shamieh 60
Said El Shamieh 61
Said El Shamieh 62
Experiment 7: Molecular identification of Staphylococcus aureus (MRSA and
MSSA)
Experiment Objective: The objective of this experiment is to be able to differ between MRSA and MSSA
using PCR
Said El Shamieh 63
Coagulase negative staphylococci have been frequently isolated as an agent of nosocomial infections in last
twenty years although they are pathogens which have low virulence rate. They are colonized in hospital
environment and hospitalized patients’ skin. Immunosuppressive therapies, invasive procedures, common
usage of broad-spectrum antibiotics tend to result in bacterial infections. NNIS (National Nosocomial
Infections Surveillance) and SCOPE studies in ICUs in USA reported that CoNS was an important agent
that may lead to hospital acquired bacteriemia EPIC (Europe Prevalence Infection Committee) study also
reported that CoNS was the fourth most common cause of nosocomial infections. CoNS oxacillin
(methicillin) resistant strains are endemic all over the world. CoNS also plays an important role such as
reservoir out of which antibiotic resistance genes might be transferred to other Staphylococcus species. All
this turns beta-lactam antibiotics ineffective for MRSA infection treatment and causes failure of therapy.
Usage of molecular techniques for detection of mecA gene in methicillin resistant bacteria is accepted
as “a gold standard”. The heterogeneous resistant strains with microorganism population can be
detected by PCR.
MRSA and mecA gene:The mecA gene is a gene found in bacterial cells which allows a bacterium to be
resistant to antibiotics such as methicillin, penicillin and other penicillin-like antibiotics. The most
commonly known carrier of the mecA gene is the bacterium known as Methicillin-resistant Staphylococcus
aureus (MRSA). In Staphylococcus species, mecA is spread on the SCCmec genetic element. resistant
strains are responsible for many infections originating in hospitals. The mecA gene does not allow the
ringlike structure of penicillin-like antibiotics to bind to the enzymes that help form the cell wall of the
bacterium (transpeptidases), and hence the bacteria is able to replicate as normal. mecA is located on the
staphylococcal chromosome cassette mec.
The objective of this experiment is to isolate high molecular weight DNA suitable for spooling.
IMPORTANT
used incorrectly.
• DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS.
The Experiment: Isolate the Bacterial DNA

To identify the bacteria, please use QIAamp DNA Blood Mini Kits !
The QIAamp procedure is suitable for use with fresh or frozen whole blood and blood which has been
treated with citrate, heparin, or EDTA. Prior separation of leukocytes is not necessary. Purification requires
no phenol/chloroform extraction or alcohol precipitation, and involves very little handling. DNA is eluted in
Buffer AE or water, ready for direct addition to PCR or other enzymatic reactions. Alternatively, it can be
safely stored a t –30 to –15°C for later use.
Said El Shamieh 64
Procedure (For more details, please check pages; 24,25)
1. Pipet 20μl QIAGEN Protease (or proteinase K) into the bottom of a 1.5ml microcentrifuge tube.
2. Add 200μl sample to the microcentrifuge tube. Use up to 200μl whole blood, plasma, serum, buffy coat,
or body fluids, or up to 5 x 106 lymphocytes in200μl PBS.
3. Add 200μl Buffer AL to the sample. Mix by pulse-vortexing for 15 s.

To ensure efficient lysis, it is essential that the sample and Buffer AL are mixed thoroughly to yield a
homogeneous solution.
4. Incubate at 56C for 10 min.
5. Briefly centrifuge the 1.5ml microcentrifuge tube to remove drops from the inside of the lid.
6. Add 200μl ethanol (96–100%) to the sample, and mix again by pulse-vortexing for 15 s. After mixing,
briefly centrifuge the 1.5ml microcentrifuge tube to remove drops from the inside of the lid.
7. Carefully apply the mixture from step 6 to the QIAamp Mini spin column (in a 2ml collection tube)
without wetting the rim. Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp
Mini spin column in a clean 2ml collection tube (provided), and discard the tube containing the filtrate.*
8. Carefully open the QIAamp Mini spin column and add 500μl Buffer AW1 without wetting the rim. Close
the cap and centrifuge at 6000xg (8000 rpm) for 1 min.
9. Carefully open the QIAamp Mini spin column and add 500μl Buffer AW2 without wetting the rim. Close
the cap and centrifuge at full speed (20,000 x g; 14,000 rpm) for 3 min.
10. Recommended: Place the QIAamp Mini spin column in a new 2ml collection tube (not provided) and
discard the old collection tube with the filtrate. Centrifuge at full speed for 1 min.
11. Place the QIAamp Mini spin column in a clean 1.5ml microcentrifuge tube (not provided), and discard
the collection tube containing the filtrate. Carefully open the QIAamp Mini spin column and add 200μl
Buffer AE or distilled water.
Module I: Performing the Polymerase Chain Reaction

Taq PCR Master Mix provides a final concentration of 1.5mM MgCl2 in the reaction mix, which will give
satisfactory results in most cases.
Reaction mix 10μl
Said El Shamieh 65
Template DNA 100ng
RNase-free water up to 20μl
IMPORTANT: After amplification, samples can be stored overnight at 2–8°C, or at –20°C for longer
storage.
PCR conditions:
• 95°C 2 min initial denaturation step
35 cycles of
• 95°C 30 sec denaturation
• 59°C 60 sec annealing
• 70°C 2 min elongation
• 70°C 5 min final elongation step
The PCR fragments obtained should be examined by electrophoresis in 1.5% agarose gel. One-kb DNA
marker needs to be used. An electric field of 100 V should applied during electrophoresis for 45 minutes.
Said El Shamieh 66
Module II: Separation of PCR Products by Electrophoresis
1. DILUTE concentrated (50X) buffer with distilled water to create 1X buffer (see Table A).
2. MIX agarose powder with 1X buffer in a 250ml flask (see Table A).
3. DISSOLVE agarose powder by boiling the solution. MICROWAVE the solution on high for 1 minute.
Carefully REMOVE the flask from the microwave and MIX by swirling the flask. Continue to HEAT the
solution in 15-second bursts until the agarose is completely dissolved (the solution should be clear like
water).
5. While agarose is cooling, add 4uL of Ethidium bromide. PLACE the well template (comb) in the
appropriate notch.
6. POUR the cooled agarose solution into the

prepared gel-casting tray. The gel should thoroughly
solidify within 20 minutes. The gel will stiffen and
become less transparent as it solidifies.
7. REMOVE end caps and comb. Take particular

care when removing the comb to prevent damage to
the wells.
8. PLACE gel (on the tray) into electrophoresis chamber. COVER the gel with 1X electrophoresis buffer
Said El Shamieh 67
(See Table B for recommended volumes). The gel should be completely submerged.
9. LOAD the entire volume (25μl) into the well.
10. PLACE safety cover. CHECK that the gel is properly oriented. Remember, the DNA samples will
migrate toward the positive (red) electrode.
11. CONNECT leads to the power source and PERFORM electrophoresis.
and proceed to STAINING the agarose gel.
background
Said El Shamieh 68
Experiment 8: VNTR Human DNA Typing Using PCR
Experiment Objective: In this experiment, students will extract their own genomic DNA. The Polymerase
Chain Reaction (PCR) and Agarose Gel Electrophoresis will be used to analyze polymorphisms between
individuals at the D1S80 region of chromosome 1.
Said El Shamieh 69
VNTR HUMAN DNA TYPING
In humans, DNA is packaged into 23 pairs of chromosomes that are inherited from an individual’s
biological parents. Although most of this genetic material is identical in every person, small
differences or “polymorphisms” in the DNA sequence occur throughout the genome, making each of us
unique. For example, the simplest difference is a Single Nucleotide Polymorphism (or SNP).
Short repetitive stretches of DNA at specific locations in the genome can vary in number to produce
STRs (Short Tandem Repeats) and longer repetitive segments are called VNTRs (Variable Number of
Tandem Repeats). Most polymorphisms occur in non-coding regions of DNA, but those that do not
may disrupt a gene and can result in disease. Medical diagnostic tests are used routinely to identify
specific polymorphisms associated with
disease.
Analyzing several different polymorphisms
within a person’s genome generates a
unique DNA “fingerprint”. DNA fingerprints
can allow us to distinguish one individual
from another. Because polymorphisms are
inherited, DNA fingerprints can also be
used to determine paternity/maternity
(and other familial relationships). The best-
known application of DNA fingerprinting is
in the field of forensic science. The first step
in forensic DNA fingerprinting is the legal
collection of biological evidence (often
present as a stain) from the crime scene or
victim. The sample is treated with a
detergent to rupture (lyse) cell membranes,
and the cellular DNA is extracted for further
analysis (Figure 1). After DNA is extracted
from these samples, forensic scientists can
develop a DNA fingerprint. The DNA
fingerprint from a crime scene can then be
compared to the DNA fingerprints of
different suspects. A match provides strong
evidence that the suspect was present at
the crime scene.
Said El Shamieh 70
The first use of forensic DNA fingerprinting occurred in the United Kingdom in 1984, following the
pioneering work of Dr. Alex Jeffreys at the
University of Leicester. Analysis by Jeffreys
led to the apprehension of a murderer in
the first DNA fingerprinting case in
September, 1987. The first conviction using
DNA evidence occurred on November 6,
1987 in Orlando, Florida. Since then, DNA
analysis has been used in thousands of
convictions. Additionally, hundreds of
convicted prison inmates have been
exonerated from their crimes, including
several death row inmates. The original
DNA fingerprinting technology utilized a
method called Restriction Fragment Length
Polymorphism (RFLP) analysis, which
involves digesting the DNA with restriction
enzymes, separating the fragments by
agarose gel electrophoresis, transferring
the DNA to a membrane, and hybridizing
the membrane with probes to polymorphic
regions. Although RFLP is very precise, it is
time-consuming and requires large
amounts of DNA. Because of this, the RFLP
method is no longer used in forensics;
however, it remains in use in certain
medical diagnostic tests. Today, forensic
scientists use the Polymerase Chain
Reaction (PCR) to produce DNA
fingerprints. PCR is a technology that has
further revolutionized the science of DNA
fingerprinting based on its ease of use and
its ability to amplify DNA. This technique
allows researchers to quickly create many
copies of a specific region of DNA in vitro.
PCR requires 500-fold less DNA than
traditional RFLP analysis and it can be
performed in one afternoon. PCR was
invented in 1984 by Dr. Kary Mullis at the
Cetus Corporation in California. For this
ground breaking technology, Mullis was
awarded the Nobel Prize in Chemistry in
1993.
Forensic scientists use PCR to analyze
highly polymorphic DNA regions. By
examining several different VNTRs or STRs
Said El Shamieh 71
from the same individual, investigators obtain a unique DNA fingerprint for that individual which is
unlike that of any other person (except for an identical twin). One VNTR, known as D1S80, is present
on human chromosome 1. It comprises a 16-nucleotide sequence that is repeated between 16 and 40
times. An individual who is homozygous for the D1S80 genotype will have equal repeat numbers on
both homologues of chromosome 1, displaying a single PCR product following agarose gel
electrophoresis (Figure 2A). More commonly, a person will be heterozygous at this locus, resulting in
differing D1S80 repeat numbers. Amplification of DNA from heterozygous individuals will result in
two distinct PCR products (Figure 2B). For most applications, law enforcement agencies will analyze
STRs, as their smaller size makes them easier to amplify, thus requiring less starting DNA.
Before performing PCR, template DNA is extracted from various biological sources (in forensic cases -
blood, tissue, or bodily fluid). Because PCR is very sensitive, only a few copies of the gene are required.
Nevertheless, freshly isolated DNA will provide better amplification results than older DNA specimens
that may have become degraded.
In order to amplify the specific DNA or target sequence, two primers (short & synthetic DNA
molecules) are designed to correspond to the ends of the target sequence.
Said El Shamieh 72
In this experiment, students will extract their own genomic DNA. The Polymerase Chain Reaction (PCR)
and Agarose Gel Electrophoresis will be used to analyze polymorphisms between individuals at the D1S80
region of chromosome 1.
LABORATORY SAFETY:
used incorrectly.
• DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS.
• Contaminated laboratory waste (saliva solution, cup, pipet, etc.) must be disinfected with 15% bleach
solution prior to disposal. Be sure to properly dispose any biological samples according to your institutional
guidelines.
thoughts and observations while conducting the experiment, and, of course, any data collected. Today, you'll
be documenting your experiment in a laboratory notebook or on a separate worksheet.

experiment.


Said El Shamieh 73
Isolation of DNA from Human Cheek Cells
Protocol: DNA Purification from Buccal Swabs using QIAamp DNA Blood Mini
Important points before starting

■ Due to the increased volume of Buffer AL that is required for the buccal swab protocol, fewer
preparations can be performed. Additional Buffer AL can be purchased separately.
■ To collect a sample, scrape the swab firmly against the inside of each cheek 6 times. Air-dry the swab for
at least 2 h after collection. Ensure that the person providing the sample has not consumed any food or drink
in the 30 min prior to sample collection.
■ All centrifugation steps are carried out at room temperature (15–25oC).
Things to do before starting
■ Prepare a 56oC water bath for use in step 3.
■ Equilibrate Buffer AE or distilled water to room temperature (15–25oC) for elution in step 10.
■ Ensure that Buffer AW1, Buffer AW2, and QIAGEN Protease have been prepared according to the
instructions.
■ If a precipitate has formed in Buffer AL, dissolve by incubating at 56oC.
Procedure
1. Place buccal swab in a 2ml microcentrifuge tube. Add 400μl (cotton and DACRON swab) or 600μl
(Omni Swab) PBS to the sample.
The Omni Swab is ejected into the microcentrifuge tube by pressing the stem end towards the swab.
Cotton or DACRON swabs are separated from the stick by hand or with scissors.
QIAamp Mini spin columns copurify RNA and DNA in parallel when both are present in the sample. RNA
may inhibit some downstream enzymatic reactions, but not PCR. If RNA-free genomic DNA is required, 4μl
Said El Shamieh 74
of an RNase A stock solution (100 mg/ml) should be added to the sample prior to the addition of Buffer AL.
2. Add 20μl QIAGEN Protease stock solution (or proteinase K) and 400μl (cotton or DACRON swab)
or 600μl (Omni Swab) Buffer AL to the sample. Mix immediately by vortexing for 15 s.
To ensure efficient lysis, it is essential that the sample and Buffer AL are mixed immediately and
thoroughly.
3. Incubate at 56oC for 10 min. Briefly centrifuge to remove drops from inside the lid.
4. Add 400μl (cotton or DACRON swab) or 600μl (Omni Swab) ethanol (96–100%) to the sample, and
mix again by vortexing. Briefly centrifuge to remove drops from inside the lid.
5. Carefully apply 700μl of the mixture from step 4 to the QIAamp Mini spin column (in a 2ml
collection tube) without wetting the rim. Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1
min. Place the QIAamp Mini spin column in a clean 2ml collection tube (provided), and discard the
tube containing the filtrate.*
Close each spin column to avoid aerosol formation during centrifugation.
6. Repeat step 5 by applying up to 700μl of the remaining mixture from step 4 to the QIAamp Mini
spin column.
Close the cap and centrifuge at 6000 x g (8000 rpm) for 1 min.
Place the QIAamp Mini spin column in a clean 2ml collection tube (provided), and discard the collection
tube containing the filtrate.*
Close the cap and centrifuge at full speed (20,000 x g; 14,000 rpm) for 3 min.
9. Recommended: Place the QIAamp Mini spin column in a new 2ml collection tube (not provided)
and discard the old collection tube with the filtrate. Centrifuge at full speed for 1 min.
This step helps to eliminate the chance of possible Buffer AW2 carryover.
10. Place the QIAamp Mini spin column in a clean 1.5ml microcentrifuge tube (not provided), and
discard the collection tube containing the filtrate. Carefully open the QIAamp Mini spin column and
add 150μl Buffer AE or distilled water. Incubate at room temperature
Said El Shamieh 75
Module II: Amplification of the D1S80 Locus
D1S80 PCR Amplification Protocol
1. Wear gloves, and use only PCR-certified sterile tubes, tips, and solutions.
Be very conscious of human contamination.
2. Set up a PCR reaction in a 200 µL PCR tube (thin-walled tube can collapse if squeezed too hard). Keep
tubes on ice until ready to place into thermocycler.
3. Prepare the PCR mix using the below concentrations:
Reaction mix 10μl
Template DNA 100ng
RNase-free water (provided) up to 20μl
5. Turn on the thermocycler.
• 95°C 4 min initial denaturation step
30 cycles of
• 95°C 30 sec denaturation
• 65°C 30 sec annealing
• 72°C 2 min elongation
• 72°C 7 min final elongation step
6. Load tubes into thermocycler (note the rack number of your tube).
Push “Run” button.
7. When PCR runs are complete, quickly place tubes on ice to stop reactions.
Prepare the agarose gel. DO NOT FORGET to add 4uL of Ethidium Bromide.
8. Run the gel at 5 V/cm (distance between electrodes) until the orange dye front is 75% to the end (100
bp band is at Orange G dye front).
Said El Shamieh 76
Module III: Separation of PCR Products by Electrophoresis
9. View stained gel on appropriate transilluminator.

Photograph gel with the appropriate camera filter.
Measure migration distance for 100-bp Ladder bands.
Measure migration distance for your D1S80 bands.
Plot standard curve for 100-bp Ladder on semilog graph paper
(log bp vs. migration distance).
Calculate size (bp) of your D1S80 bands (subtracting flanking sequence).
Determine number of VNTR repeats in each allele.
Said El Shamieh 77
Study Questions
1. Compare your D1S80 PCR product with those of the rest of the class. Did any students have genotypes
similar to yours? How could you explain such similarities?
2. What is polymorphic DNA? How is it used for identification purposes?
3. What is CODIS? How is it used to solve crimes?
4. What is the difference between a STR and a VNTR? Which (STR or VNTR) is predominantly used in law
enforcement?
Said El Shamieh 78
Experiment 9: Principles of DNA Sequencing
Experiment Objective: The objective of this experiment is to develop an understanding of DNA
sequencing and analysis. This is a dry lab which contains autoradiographs from an actual DNA sequencing
experiment.
Said El Shamieh 79
DNA SEQUENCING
The basic unit of all living organisms, from bacteria to humans, is the cell.
Contained within each cell is a molecule called deoxyribonucleic acid (or DNA). Today, we know that DNA
is the blueprint used to build an organism – our genetic makeup, or genotype, controls our observable
characteristics, or phenotype. The directions encoded in our genes control everything from growth and
development to cell-type specification, neuronal function, and metabolism.
A strand of DNA is composed of building blocks known as deoxynucleotides
(dNTPs) that are linked together into a long chain (Figure 1). Each dNTP
comprises three basic parts: a phosphate group, a deoxyribose sugar, and a
nitrogen-containing base. While the phosphate group and deoxyribose
sugars are constant, the specific bases determine the identity of the dNTP:
adenine (A), cytosine (C), guanine (G), or thymine (T). The order of these
nucleotides in the chain gives rise to genes, each with a unique sequence.
In addition, the 3’ hydroxyl group on the sugar of one nucleotide forms a
covalent bond with the 5’ phosphate group of its neighbor, resulting in DNA
strands with a distinct polarity. This polarity ensures that the strand of DNA
is read in the correct
direction during gene
transcription.
DNA sequencing is the
process of determining the
nucleotide order for a DNA
segment of interest. As
recently as the 1970s,
scientists were unable to
sequence even the most
simple of genomes. This all
changed in 1977 when Fred Sanger and his team exploited
the natural process of DNA replication to allow scientists to
sequence DNA accurately. This method, now known as
“Sanger Sequencing”, resulted in the 1980
Nobel Prize in Chemistry and is still in use today.
During Sanger Sequencing, an enzymatic reaction is
performed to create a new DNA sequence that is
complementary to the target or template DNA. This
reaction requires
DNA polymerase (the enzyme that will create DNA molecules by assembling nucleotides), a short DNA
primer that serves as a starting point for sequencing, and two types of nucleotides – normal dNTPs and a
specialized base analog called a dideoxynucleotide (ddNTP). The ddNTPs lack a hydroxyl-group at the 3’
carbon of the ribose sugar, which prevents new nucleotides from being added to the DNA strand after a
ddNTP has been incorporated (Figure 2).
Said El Shamieh 80
DNA synthesis is initiated where the
primer is annealed to the template. The
polymerase then adds nucleotides to the
strand in an order that complements the
original single stranded template, always
pairing C with G and A with
T. This process continues to construct a
DNA fragment until a ddNTP is
incorporated, at which point no other
nucleotide can be added.
The initial ddNTP concentration in the
reaction is kept relatively low so that they
are incorporated into a growing DNA
strand randomly and infrequently. The
result is the generation of millions of DNA
fragments with different end points (Figure
3).
During a sequencing experiment four
separate enzymatic reactions are
performed, one for each ddNTP. For
example, the “G” reaction contains
dideoxyGTP, while the “C” reaction contains dideoxyCTP. As the sequencing experiment progresses the
ddNTPs will be randomly added to terminate the growing chain. Since the reaction contains only one type of
ddNTP, each reaction will contain a population of molecules with the same 5’ start site and with the same
ddNTP base at the 3’ end. However, since the ddNTP is inserted randomly the reaction will contain
nucleotide fragments of different sizes, depending on when the ddNTP was incorporated (Figure 3).
The next step in a sequencing reaction involves using polyacrylamide gel electrophoresis to analyze the
sequencing fragments. First, the sequencing reactions are added into depressions (or “wells”) within a
polyacrylamide gel and an electrical current is passed through the gel. Because the sugar-phosphate
backbone of DNA has a strong negative charge, the current drives the DNA through the gel towards the
positive electrode. At first glance, a polyacrylamide gel appears to be a solid at room temperature. However,
on the molecular level, the gel contains small channels through which the DNA can pass. Small DNA
fragments move through these holes easily, but large DNA fragments have a more difficult time squeezing
through the tunnels. Because molecules of different sizes travel at different speeds, they become separated
and form discrete “bands” within the gel. This technique is precise enough to distinguish fragments that
differ in size by a single nucleotide. When used after sequencing, gel electrophoresis of the four ddNTP
reactions can reveal the DNA’s sequence, with each fragment corresponding to a different nucleotide
position.
While polyacrylamide gel electrophoresis can separate the DNA fragments, the DNA bands are initially
clear and colorless. To visualize the DNA, scientists add radioactive dATP into the four reactions, allowing
it to incorporate into the DNA strands. After the electrophoresis is completed, the radiolabeled DNA can be
visualized by autoradiography.
The polyacrylamide gel is placed into contact with a sheet of x-ray film, allowing the DNA fragments to
create a dark exposure band on the film. Since the smallest fragments move through the gel faster than the
larger fragments, the sequence is read from the bottom of the gel to the top. For example, the simulated
autoradiograph (or “autorad”) in Figure 4 would result from Sanger sequencing analysis.
DNA sequencing allows us to understand the kind of genetic information that a segment of DNA carries.
Said El Shamieh 81
For example, in sections of DNA that code for a protein the nucleotide order determines amino acid order.
This in turn determines the type of protein produced by a cell. Because of this, sequence information can be
used to analyze genes for changes, know as mutations. Sequencing has become essential for many different
scientific disciplines. For example, molecular biologists and geneticists use sequencing information to
explore how our genes affect cellular behavior. Similarly, evolutionary biologists can use sequence
information to provide clues about similarities and differences between species. Finally, doctors and
researchers use DNA sequencing to diagnose harmful mutations and provide counseling and preventative
care for diseases.
In the laboratory, scientists identify mutations by

comparing their sequence information to a reference
sequence that represents the “wild type”, or established,
sequence of the gene (summarized in Figure 5). One
common type of mutation is called a substitution
mutation. In a substitution mutation one base pair
nucleotide is replaced by another base pair nucleotide –
for example a T replaces a G. Many times, the mutation is
silent, meaning that the mutation is not harmful to the
organism. When the sequence is in a coding region such a
change can alter the protein product by changing one
amino acid in the protein sequence (missense) or by
creating a new stop signal that ends the creation of that
protein (nonsense).
Two other types of mutations are insertion and deletion mutations. An insertion mutation changes the
number of DNA bases by adding a piece of DNA,
whereas a deletion mutation changes the number of
DNA bases by removing a piece of DNA. These
insertions or deletions, known collectively as
"indels", can be as small as one base pair or as large
as an entire chromosome region. Even a one base
pair indel can cause dramatic changes when it
occurs in a protein-coding region. For example, if an
insertion occurs and the number of inserted
nucleotides is not a multiple of three then every
amino acid following the insertion will be different.
The same is true for deletions. This type of change,
where the grouping of the nucleotides is changed, is
known as a frame shift mutation.
In this experiment, you will read the autoradiograph
of DNA sequenced from the bacteria virus M13.
The nucleotide sequence is then compared with a
reference sequence to identify any mutations.
Said El Shamieh 82
Experiment Procedures
The objective of this experiment is to develop an understanding of DNA sequencing and analysis. This is a
dry lab which contains autoradiographs from an actual DNA sequencing experiment.
EXPERIMENT PROCEDURE:
1. Obtain the sample autoradiograph and place it on a light box to enhance visualization.
2. The sequencing reactions have all been loaded in order: G-A-T-C.
3. Begin analysis of the DNA sequence at the bottom of the autoradiograph with the circled band, which is
an A.
4. Compare the deduced sequence to the wild type sequence shown in Figure 6.
5. Identify the location of the mutant nucleotide. What was the mutation? Is there more than one mutation?
Study Questions
1. Why is DNA important? What is it made of?
2. What is DNA Sequencing?
3. Why are Sanger sequencing experiments divided into four reactions?
4. Name and describe five reagents needed to carry out Sanger sequencing.
Said El Shamieh 83
Experiment 10: Human Karyotype
Experiment Objective: the objective of this experiment is to use the karyotyping techniques for diagnosing
a chromosomal disorder.
Said El Shamieh 84
Cytogenetics
It is the study of genetic events at the cell level, that is to say at chromosomes without the need to extract
DNA: chromosomal abnormalities (number and structure), recombination of chromosomes, etc. The
techniques used are mainly the realization of karyotype (karyotyping), methods of FISH (Fluorescent In-Situ
Hybridization: hybridization in situ by fluorescent probes), using DNA chip (DNA-microarray, biochip ").
Cytogenetics allow the evaluation of the chromosomal composition of an individual (the composition of
chromosomes of an individual cell). For example, a normal man is 46, XY, that is to say that he has:
◊ 46 chromosomes per cell (23 pairs)
◊ 2 gonosomes (X and Y are the two sex chromosomes) and 44 autosomes.
Eukaryotes have multiple linear chromosomes contained in the cell nucleus. Each has its own chromosome
centromere, with one or two arms (p and q) projecting from it.
During mitosis and meiosis, the centromere allows the assembly of the kinetochore that links chromosomes
to microtubules, thus allowing their movement and distribution between the two daughter cells.
The chromosome ends are special structures called telomeres. These ends shorten at each replication
because DNA polymerase requires a primer to start replication. An enzyme, telomerase, can in some cases
restore the length of the telomere. DNA replication starts at various locations of the chromosome.
Eukaryotic chromosomes can be distinguished according to their

centromeric position:
• It is a metacentric chromosome (or centromeric) when the
centromere in the central position (middle position), which gives
arms (or chromatids) of approximately equal length.
• It is a submetacentric chromosome when the centromere is almost centro
in the central position, which gives arms (chromatids) unequal in
length (a small arm "p" and a long arm 'q').
• It is an acrocentric chromosome when the centromere is more
closer to one of the two ends (telomeres).
• It is a telocentric chromosome when the

centromere is very close to the chromomal
telomeres.
• It is an acentric chromosome when the
centromere is lost (anomaly).
Other abnormalities may cause the appearance of a chromosome with two centromeres or dicentric
chromosome; it is unstable and may break (during meiosis) into different segments which are distributed
randomly in each of the daughter cells.
Techniques
Said El Shamieh 85
The first technique of cytogenetics is the achievement of karyotypes / caryogram (karyotyping), where the
chromosomes are usually stained with Giemsa stain. (Then, another techniques appeared: FISH
(Fluorescent In Situ Hybridization), allowing a much better resolution in a shorter time.
Karyotyping technique:
It is a conventional technique of postnatal constitutional karyotyping.
• The karyotype allows the observation and classification of chromosomes during metaphase or
prometaphase of mitosis.
• The resolution of conventional cytogenetics is that of light microscopy.
Indications for carrying out the karyotyping experiment: It is carried out in the following situations:
•-mental retardation or developmental delay;
•-dysmorphic features;
•-Congenital malformation;
•-Sexual ambiguity;
•-A family history of chromosomal abnormality;
•-Recurrent miscarriages;
•-Fertility problems
•-Preliminary screening for in vitro fertilization (gametes).
Applications of karytoyping: Karyotyping is used to confirm the diagnosis of diseases caused by

chromosomal aberrations which may include changes in the number of chromosomes, such as trisomy
(three copies of a chromosome), monosomy (one copy of a chromosome) or triploidy (three copies of each
chromosome that carries the total to 69). Translocation is another form of chromosomal aberration that can
be determined by karyotyping. Translocation is the transfer of a fragment of a chromosome to another
chromosome. The end of one chromosome may detach and join to the end of another chromosome, often
where it is lacking an end. Since it is not always easy to detect the translocation, we often use the
analysis of fluorescence in situ hybridization to confirm a translocation perceived in a karyotype.
ESTABLISHMENT OF KARYOTYPE (actual experiment).
A-Obtaining dividing cells:

• The blood lymphocytes are most often used: whole blood collected under sterile heparin, is incubated
from 48 to 72 hours in culture medium (in vitro) containing a high mitogenic lectin
(phytohaemagglutinin, or PHA).
• The fibroblast cells require culturing for one to three weeks. All tissues rich in fibroblasts, such as skin,
may be used. The tissue fragment is collected in a sterile culture medium containing antibiotics.
• Also, other tissues used for the realization of a human karyotype are:
-Bone marrow: hematopoietic cells
-Amniotic fluid (amniocentesis: before birth)
-Connective tissue
-Tumor tissue
B- Obtaining many metaphase cells and with good quality:

After accumulation of metaphase or prometaphase cells by synchronizing cultures and / or blocking of the
spindle apparatus; they are blocked (by a solution of colchicine or colcemid) when cell division has reached
the metaphase stage. At this stage, the chromosomes are aligned in the middle of the cell, ready to be
divided to form two new daughter cells. At this stage, the chromosomes are very condensed and can
easily be identified under a light microscope.
Said El Shamieh 86
C-Dispersion of chromosomes in the cytoplasm:
The mitotic cells are harvested, then swelled and burst by incubation in a hypotonic medium (distilled
water) to separate chromosomes (metaphase cells have no nuclear membrane); then we put the cells with a
fixative (a mixture of methanol and acetic acid) , and spread the fixed cell suspension on a glass slide for
microscope observation, this is what is known as a chromosomal spread. [methanol and acetic acid are to
dehydrate and flatten the nuclei, or paraformaldehyde (which maintains the shape of the nucleus). These
products are used depending on what we want to observe in the nucleus.
D-Staining the chromosomes:

Prior to adding the staining solution, the chromosomes must first be treated with trypsin, which will begin
to digest the chromosomes (enzymatic denaturation), enabling them to better receive the giemsa. Giemsa
(4%, for 20-25 min) is the most classic stain that results, after the application of appropriate treatment, in the
appearance of dark and light alternating bands on the chromosomes: This is what is called the G
banding (the "G-band"). The topography of the bands is characteristic of a chromosome and the
chromosome can be identified by it (both chromosomes of a pair have the same topography of bands).
The Giemsa stain consists of a mixture of two dyes [methylene blue (basic) and purplish pink eosin
(acid;) in 1902, Gustav Giemsa developed this staining technique to identify the malaria parasite
Plasmodium falciparum in blood smears].
There are mainly two types of staining:
• Giemsa staining by enzymatice denaturation (or GTG banding) shows bands called G-bands. The
Giemsa stain creates chromosomal banding patterns with the dark areas rich in adenine and thymine (A +
T; heterochromatic). The light areas are rich in guanine and cytosine (G + C), are euchromatic and early
replicating. Euchromatic DNA is a genetically active area is that stains very lightly with dye treatments.
• Giemsa staining by thermal denaturation (RHG banding for banding using Reverse Heat and Giemsa)
shows bands called R-bands. In Reverse-bands (or R-bands), the dark areas are rich in guanine and
cytosine (G + C).
Regions (active)
G-Bands R-Bands
Rich in AT Rich in GC
Poor in genes Rich in genes
Late Replication Early Replication
DNase insensitive DNase sensitive
E: After staining, microscopic observation at high magnification

F: Construction of karyotype: Mitoses photographed and printed on paper and the chromosomes are cut out
and grouped in pairs.
Said El Shamieh 87
B-Classification of the chromosomes
The normal human karyotype is written 46, XX (female) or 46, XY (male).
The simple morphology, used until the early 1970s, classifies chromosomes by their size decrease from 1
to 22 and in groups A to G. This classification of chromosomes by size and by centromeric index is very
approximate.
Labeling techniques identify each chromosome pair by their pattern of light and dark bands. The bands are
listed in an international nomenclature. Each chromosomal arm is divided, depending on its size, into
one to four regions and each region into numbered bands from the centromere to telomere. For
example, 17p12 refers to chromosome 17, short arm (p), region 1, band 2.
Based on their size, centromere position and positions of the different bands, the chromosomes are
classified into 7 groups/classes:
Group Chromosomes Description

A 1–3 Largest; 1 and 3 are metacentric but 2 is submetacentric
B 4,5 Large; submetacentric with two arms very different in size
C 6–12,X Medium size; submetacentric
D 13–15 Medium size; acrocentric with satellites
E 16–18 Small; 16 is metacentric but 17 and 18 are submetacentric
F 19,20 Small; metacentric
G 21,22,Y Small; acrocentric, with satellites on 21 and 22 but not on the Y
Autosomes are numbered from largest to smallest, except that chromosome 21 is smaller than chromosome 22.
Said El Shamieh 88
Study question
Said El Shamieh 89
1- Cite 3 Human disorders due to chromosome alterations in autosomes, then explain them at the molecular
level.
2- Identify chromosome 1, 4, 21 , 22 in the following chromosome spreading.
Said El Shamieh 90

Genetics Lab Manual

Uploaded by

Copyright:

Available Formats

Genetics Lab Manual

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Genetics Lab Manual

Uploaded by

Copyright:

Available Formats

Beirut Arab University

Faculty of Health Sciences

GENETICS AND MOLECULAR BIOLOGY LABORATORY (MELS401)

LABORATORY MANUAL (2018-2019)

Said El Shamieh, MLT| PhD.

Experiment 1: Micro-pipetting Basics

Experiment 2: Modeling DNA Structure

Experiment 3: DNA extraction and quantification

Experiment 4: Size Determination of DNA Fragments

Experiment 5: PCR Amplification of DNA

Experiment 6: Design PCR primers

Experiment 7: Identification of Staphylococcus aureus (MRSA and MSSA) using PCR

Experiment 8: VNTR Human DNA Typing Using PCR

Experiment 9: Principles of DNA Sequencing

Experiment 10: Human Karyotype

ACCURACY AND PRECISION IN BIOTECHNOLOGY

Before starting the Experiment:

During the Experiment:

After the Experiment:

1. Perform the following conversions:

In decimals In scientific notation

1ml = ____________ liter 1ml = ____________ liter

1 liter = ____________ml 1 liter = ____________ml

1ml = ____________μl 1ml = ____________μl

1μl = ____________ml 1μl = ____________ml

10μl = ____________ml 10μl = ____________ml

20μl = ____________ml 20μl = ____________ml

50μl = ____________ml 50μl = ____________ml

100μl = ____________ml 100μl = ____________ml

2. How many times greater is aml than aμl? ______________

3. How many times greater is a liter than aml? ______________

4. How many times greater is a liter than aμl? ______________

1. SET the micropipet to the appropriate

The Work of Erwin Chargaff

Before starting the Experiment:

During the Experiment:

After the Experiment:

Purification of DNA from forensic and human identity samples

Principle and procedure QIAamp

Purification on QIAamp Mini spin columns

Removal of residual contaminants

Elution of pure nucleic acids

QIAGEN Protease stock solution (store at 2–8C)

Buffer AL† (store at room temperature, 15–25C

Buffer AW1† (store at room temperature, 15–25C

Buffer AW2* (store at room temperature, 15–25C

Protocol: DNA Purification from Blood or Body Fluids

Important points before starting

3. Add 200μl Buffer AL to the sample. Mix by pulse-vortexing for 15 s.

4. Incubate at 56C for 10 min.

Before starting the Experiment:

During the Experiment:

After the Experiment:

3. DISSOLVE agarose powder by

5. While agarose is cooling, add 4 uL of Ethidium bromide dye.

RUNNING THE GEL

12. TRANSFER the gel to the UV Lamp.

1. Measure and Record Migration Distances

3. Determine the length of each unknown fragment.

c. Repeat for each fragment in your unknown sample.

1ml = liter 1ml = liter

1 liter = ml 1 liter = ml

1ml = μl 1ml = μl

1μl = ml 1μl = ml

10μl = ml 10μl = ml

20μl = ml 20μl = ml

50μl = ml 50μl = ml

100μl = ml 100μl = ml