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Techniques used in Molecular Biology

Method · December 2017

DOI: 10.13140/RG.2.2.20054.55366

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 DNA Fingerprinting
• Unless they are identical twins, individuals have
unique DNA

• DNA fingerprinting
– The name used for the unambiguous identifying
technique that takes advantage of differences in DNA
sequence

• The process of DNA fingerprinting begins by

isolating DNA from
– blood, semen, vaginal fluids, hair roots, skin, skeletal
remains, or elsewhere
1
 Polymerase Chain Reaction (PCR)
• If there is only a small amount of DNA available for DNA
Fingerprinting
– augment the amount of DNA using a technique called PCR
– PCR is doing DNA replication in a test tube

2
 Polymerase Chain Reaction (PCR)

• Like ALL DNA

5’ 3’
polymerases 3’ 5’
and cool
• Taq polymerase can to anneal

only add to the 3’ end Template

of an existing Primer
nucleotide Primer
Template

•A DNA primer that is

complementary to the
template is used to
supply that 3’ end

3
 DNA Fingerprinting
• After we isolate the DNA and amplify it with PCR
• Treat the DNA with restriction enzymes
– cut DNA at specific sequences
– Everyone’s DNA is different, so everyone’s DNA will cut at different
sites

• This results in different sized fragments

• The different sized fragments are called restriction
fragment length polymorphisms, or RFLPs
• We can observe the different sized fragments in an
experiment that separates DNA based on fragment size
called Gel Electrophoresis

4
 RFLP Analysis

• Everyone has genetic

sequences called
variable number
tandem repeats, or
VNTRs
– Everyone has different
amounts of VNTRs
– The VNTRs make the
different sized RFLPs

5
 Gel Electrophoresis

• Fragments of DNA from restriction enzyme cleavage

are separated from each other when they migrate
through a support called an agarose gel
– It is similar to the yummy food Jell-O gelatin
– It is actually made out of some of the same ingredients

• The size-based separation of Molecules of DNA

separate based on size when an electric current is
applied to an agarose gel
– This is gel electrophoresis

6
Gel Electrophoresis

7
 Gel Electrophoresis
• The separated DNA fragments are then drawn out of
the gel using a nylon membrane
• The nylon membrane is treated with chemicals that
break the hydrogen bonds in DNA and separate the
strands
• The single stranded DNA is cross linked to the nylon
membrane
– By heat or UV light
• Incubate the nylon membrane with a radioactive
probe of single stranded DNA complementary to the
VNTRs
8
Gel Electrophoresis

9
 Gel Electrophoresis
• The radioactive probe shows up on photographic
film
– Because as it decays it gives off light
– The light leaves a dark spot on the film

• Different individuals have different patterns of bands

– these make up the fingerprint

10
 Gel Electrophoresis

This Protocol is known as Southern Blotting

11
Southern Blotting
 DNA Fingerprinting
• DNA fingerprints can be used to determine which
bone fragments belong to which individual

13
 DNA Fingerprinting
• DNA fingerprints of children should be similar to the those of
parents
• DNA fingerprinting can show which individuals are the
parents of specific children

14
 Northern Blot Analysis
• Northern blotting analyzes RNA much the same way
that Southern blotting does DNA:
– RNA is extracted from the cell, undergoes gel
electrophoresis, and is bound to a filter.
– Hybridization between bound cellular RNA and a labeled
probe occurs. The sizes of the RNA fragments detected by
the probe can be determined

15
 Northern Blot Analysis

• Northern blot analysis is used for determining:

– The size(s) of mRNA encoded by a gene. Northern
blots have shown that different mRNA species arise
from the same region of DNA, suggesting differential
use of promoters and terminators, and/or alternative
mRNA processing.
– Whether a specific mRNA is present in a cell type,
and if so, at what levels. Gene activity is measured in
this way, and RNA sampling is widely used to study
development, tissue specialization, or the response of
cells to various physiological stimuli.
16
 Producing Recombinant Proteins

• The first step in the production of rBGH protein (or insulin) is

– to transfer the BGH gene (or human insulin gene) from the nucleus
of a cow cell (or human cell) into a bacterial cell

• How do we do that????
• 3 steps are involved in turning a cow BGH gene into a
recombinant BGH (rBGH) gene in a bacterial cell
– rBGH gene means that this product is genetically engineered
– with the r indicating recombinant

17
 Producing Recombinant Proteins
• 5 steps are involved in turning a cow BGH gene into a
recombinant BGH (rBGH) gene in a bacterial cell
1. Make lots of copies of the cow BGH gene in the lab in a
test tube
2. Cut cow BGH gene with restriction enzymes
3. Insert this cow BGH gene into bacterial DNA = rBGH
4. Inject the bacterial DNA containing the rBGH into bacteria
5. Grow up lots of these genetically engineered bacteria and
purify the rBGH cow protein they are making

18
 Cloning a Gene Using Bacteria

Step 1. Make lots of copies of the BGH Gene

• Use PCR to amplify only the cow BGH gene from

the cow chromosomes

• Remember, PCR is just replicating DNA in the

laboratory in a test tube

• End up with lots and lots of copies of the cow BGH

gene DNA in a test tube

19
 Cloning a Gene Using Bacteria
Step 2. Prepare the cow BGH Gene for inserting into
bacterial DNA
• The cow BGH gene ends are sliced using
restriction enzymes
• Restriction enzymes cut DNA only at specific
sequences that leave the double-stranded DNA
jagged or “sticky” on the ends

20
Restriction enzymes cut the DNA in a staggered
pattern, leaving “sticky ends”…

Restriction enzyme cut

21
 Fig. 16.2
Examples of how
different
restriction
enzymes cleave
DNA

22
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
 Cloning a Gene Using Bacteria
Step 3. Insert the BGH Gene into the Bacterial
Plasmid
• The bacterial plasmid is also cut with the
restriction enzyme, leaving sticky ends
– A plasmid is a small circular DNA that is separate from
the bacterial genome

• Sticky ends of the cut BGH DNA attach by

complementary base pairing to the sticky ends of
the cut plasmid DNA
• This is now recombinant DNA

23
 Fig. 16.3
Cleavage of DNA by the restriction enzyme EcoRI

24
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
 Cloning a Gene Using Bacteria
Step 4. Insert the Recombinant Plasmid into a Bacterial Cell
• The recombinant plasmid containing the rBGH is then placed into
bacterial cells

Step 5. Grow up bacteria and purify rBGH protein

• Large numbers of these rBGH plasmids are copied in each bacteria and
each bacteria copies itself
– Can grow up billions and billions of bacteria in the lab
– All containing the rBGH plasmid
• Each plasmid will generate rBGH protein through transcription and
translation
• Isolate the BGH protein from the bacterial proteins
• TaDa! Recombinant proteins

25
 Cloning a Gene Using Bacteria

• How can bacteria produce the cow BGH protein?

• This works because bacteria use the same genetic
code as cows (and all living things)
• Other proteins are made in this way:
– Insulin for diabetics
– Clotting factors for hemophiliacs
– Cancer treatment drugs

26
Cloning a Gene
Using Bacteria

27
 Using the lacZ gene as a reporter of gene
expression
• Reporter gene – protein encoding gene whose
expression in the cell is quantifiable by techniques of
protein detection.

• Fusion of reporter gene to cis acting (DNA)

regulatory regions (like promoters) allows
assessment gene activity by monitoring amount of
reporter gene product

28
/42
Fusion used to perform genetic studies of the regulatory region
of gene X

29
/42
Fig. 16.18 a
 Cloning is Genetic Engineering
• Cloning is the making of entire organisms using
genetic engineering

• Has been done in cattle, goats, mice, cats, pigs,

rabbits, and sheep

• Has never been done in humans

30
 Cloning is Genetic Engineering
• Dolly the sheep was the first animal to be cloned
• The DNA (all 46 chromosomes) from an adult sheep mammary gland
were fused with an unfertilized egg cell without any DNA inside
• The treated egg was placed in the uterus of an adult sheep
– that had received hormone treatments to support pregnancy

• There were 277 failures before this nuclear transfer technique

succeeded
– Dolly was successfully created in 1997

31
 Cloning is
Genetic
Engineering

32
 Cloning is Genetic Engineering

• Dolly was put to sleep at the age of 6 in 2003

– Because of health problems
– She was suffering from arthritis and a progressive lung disease
– These are usually only seen in old sheep

• Cloned animals seem to age prematurely and show signs of

other health problems that are normally associated with
aging
• Hypothesis:
– Egg and Sperm DNA is “reprogrammed” and does not reflect the age of the
parents
– Adult donor DNA is not “reprogrammed” in the egg and reflects the age of the
donor
– Using adult DNA to create new organisms results in organisms that remain at
the age of their donor DNA at birth
33
 Therapeutic Cloning
• Not cloning of entire organisms, but cloning of specific
tissues or cells
– Pancreatic cells to produce insulin in diabetics
– Spinal cord cells in parallized patients

• Stem cells are induced in the laboratory to turn into specific

tissue cells
• What are Stem Cells?
– Cells that can be induced to turn into every type of cell in the human
body

• Where are Stem Cells found?

– In embryos
– The original fertilized egg grows into an entire human being.
– These cells can make every cell type

34
 Embryonic Stem Cells

http://sps.k12.ar.us/massengale/images/ivf.jpg
35
 Stem Cells
• How do scientists acquire Stem Cells?
• Human eggs are fertilized by human sperm in vitro
– (in a test tube)

• Fertilized egg grows and divides by mitosis to an

embryo which is just a ball of 8 cells at this point
– These cells can now be frozen as stem cells for research
–
• This is the same process that is used by couples
that cannot conceive a baby
– Called in vitro fertilization (IVF)
36
 IVF
• in vitro fertilization (IVF)
– The ball of 8 cells is implanted into the female’s uterus
– She has been treated with hormones to simulate pregnancy to accept
the embryo

http://pregnancyandbaby.com/pregnancy/baby/Facing-infertility-5476.htm 37
 Stem Cells
• Couples using IVF generally generate 15-30 frozen embryos and use
only 3-9 of them
• The remaining embryos can either be thrown away or donated to stem
cell research
• Stem cells can NEVER be acquired by abortion or miscarriage
– There are no embryonic stem cells left, they have already changed
– The 8 cell stage is before implantation in the uterus
– Before anyone could even know they have conceived

• Stem cell researchers use more donated embryos rather than ones
created in the laboratory specifically for research
• Most stem cell research uses embryonic stem cell lines
– Cells that originally came from an 8 cell embryo, but have been manipulated in the lab
to continue growing as separate cells in a flask
– They do not form any tissues, they just grow as individual stem cells
– Researchers can grow millions and millions of these in the lab to perform studies that
may someday save lives and cure diseases
38
8 Cell Stage
Embryonic
Stem Cells

39
 Stem Cells
• The use of embryonic stem cells in research fuels a
heated national debate
– Mostly because of scientific ignorance
• Embryonic stem cells are valued by researchers
because they are totipotent, or able to become any
other cell
– With increased study, these could potentially treat or cure
any type of disease and cancer
• In 2001, President Bush banned federal funding for
reaching using embryonic stem cells
– Because he never took Bio 360!!!
– (or any biology for that matter)
40

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