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Lecture 3-DNA Analysis I

The lecture covers DNA analysis techniques, focusing on Allele-Specific Oligonucleotide (ASO) hybridization and Polymerase Chain Reaction (PCR). ASO is used for detecting mutations, while PCR allows for selective amplification of DNA, with applications in disease detection and forensic analysis. The lecture also discusses the importance of primer design and introduces quantitative PCR (qPCR) for measuring DNA quantities during amplification.

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Ahmed Ali
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2 views

Lecture 3-DNA Analysis I

The lecture covers DNA analysis techniques, focusing on Allele-Specific Oligonucleotide (ASO) hybridization and Polymerase Chain Reaction (PCR). ASO is used for detecting mutations, while PCR allows for selective amplification of DNA, with applications in disease detection and forensic analysis. The lecture also discusses the importance of primer design and introduces quantitative PCR (qPCR) for measuring DNA quantities during amplification.

Uploaded by

Ahmed Ali
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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Week 3-Lecture 3

DNA Analysis Part I

Presented by
Dr.Ibrahim Ashraf Abdelwahab
Lecturer of Microbiology and Immunology dept.,
Faculty of Pharmacy, Pharos University In Alexandria.
Competencies & Key elements achieved by learning outcomes of the
Lecture:
Competency Key elements Learning Outcomes
2-3- 2-3-1 Handle, identify, and dispose 1. Distinguish between different assays used in
Handle and dispose biologicals biologicals, synthetic/natural materials, the microbiological quality control of
and synthetic/natural biotechnology-based and radio-labeled pharmaceutical products.
pharmaceutical products, and other materials/products used 2. Outline the different serological and molecular
materials/products effectively and in pharmaceutical field. methods used to identify microorganisms.
safely with respect to relevant
laws and legislations.
2-5- 2-5-3 Contribute in planning and conducting 3. Determine the appropriate serological and
Contribute in pharmaceutical research studies using appropriate molecular methods used in research studies.
research studies and clinical methodologies.
trials needed to authorize
medicinal products.
3-1-Apply the principles of 3-1-2 Apply the principles of public health 4. Recognize the range of advanced laboratory
body functions to participate and pharmaceutical microbiology to select and important molecular and serological
in improving health care and assess proper methods of infection techniques.
services using evidence- control. 5. Determine the different serological and
based data. molecular methods used to identify
microorganisms and laboratory safety measures
3-1-4 Relate etiology, epidemiology, to prevent and control the effects of industrial
pathophysiology, laboratory diagnosis, and and laboratory hazards/accidents.
clinical features of infections/diseases and
their pharmacotherapeutic approaches. 6. Identify principles of various tools and
instruments, and select the proper techniques
for synthesizing and analyzing different
biological materials.
4-2- 4-2-2 Use contemporary technologies and media 9. Compile information from a variety of sources
Effectively communicate skills. (library, electronic, and online resources) to
verbally, non-verbally and in accomplish required assignments
writing with individuals and
communities.
1. Allele-Specific Oligonucleotide
Hybridization
❖Allele-specific oligonucleotide (ASO) hybridization, or
dot blot analysis, can detect specific mutations in
particular disorders. This assay is based on the
principle that when probing a region of DNA, even a
single-base-pair change between a target region and
the probe can destabilize the hybrid. In general, two
synthetically created probes are designed for the area
of interest, one complementary to the wild-type
allele, and one complementary to the mutant allele.
The digested DNA is separated by gel electrophoresis and
immobilized on a membrane. It is then hybridized with
radioactively labeled probes. If both probes react, then
the individual is heterozygous for the mutation of
interest. If only one probe reacts, then that individual is
homozygous for either the wild-type or mutant allele.

This technique was used to detect the sickle-cell allele


and prenatal diagnosis of β-thalassemia.

ASO was also a technique that benefitted from the use of


PCR. Instead of probing non-amplified genomic or cloned
DNA, regions of interest could be PCR amplified first and
then probed. Adding PCR to ASO allowed for a more
rapid detection of mutations.
1. Allele-Specific Oligonucleotide
Hybridization
2-PCR
Polymerase Chain Reaction
❖ selective amplification of a
chosen region of DNA molecule
that has known sequences at its
borders to act as primers
❖PCR application :
a) obtain a pure sample of a gene
without the need for a selection
problem in cloning
b) Detection of disease if its gene
primer is known
c) Test for the presence of
mutations eg; thalassaemia.
human globin genes are known.
PCR
❖PCR Advantages :
➢ PCR is tremendously sensitive: can detect even just
ONE DNA molecule in the sample
✓ Detection of infection early
✓ Forensic material
➢ Gene is automatically “selected” as a result of the
positions at which the primers anneal.
➢ Replication in an exponential pattern
❖PCR has two limitations:
➢Sequences of primers must be known for easy
experimentation
but if unknown → cloning first is a must
➢ The length of the DNA sequence, to be copied, is limited.
Five (kb) → copied easily
up to forty kb → difficult
BUT many genes, humans and other vertebrates, longer
than this.
SO Cloning first is a must.
❖Designing the oligonucleotide primers for a PCR
❑ The primers are the key to the success or failure of a
PCR experiment
If correct → amplification of a single targeted DNA
fragment
Incorrect →no amplification or wrong fragment
amplification, or more than one
fragment amplified
❑Length of primer: 17-mer length is best
if too short 8 mer → might hybridize to non-target
sites.
too long 30 mer → hybridization at a slower rate
→lower PCR efficacy.
[ The efficiency of the PCR: number of amplified molecules
during experiment ]
❖DNA–DNA hybridization is a temperature-dependent
phenomenon :
❑Melting temperature ( Tm ): the temperature at which
the correctly base-paired hybrid dissociates (“melts”).
Tm = (4 × [G + C]) + (2 × [A + T])°C
[G + C] is the number of G and C nucleotides in the primer
[A + T] is the number of A and T nucleotides.
❑The two primers should be designed so that they have
identical Tms.
❑Optimum temperature < Tm by 1 or 2 degrees
If too high → no hybridization takes place bet
primers and templates
too low → some mismatched hybrids
after PCR
I. Gel electrophoresis of PCR products
II. Sequencing of PCR product
I. Gel electrophoresis of PCR products
1) determine if a PCR experiment has worked
2) presence of restriction sites in the amplified region
of the template DNA by treating the PCR product
with a restriction endonuclease before agarose gel.
This is a type of restriction fragment length
polymorphism (RFLP) analysis is important in the
construction of genome maps and in studying
genetic diseases
3. establish if the template DNA contains an insertion or
deletion mutation in the amplified region. Length
mutations of this type form the basis of DNA profiling, a
central technique in forensic science.
Quantitative PCR (qPCR):
• Quantitative PCR (qPCR): the amount of product
synthesized during a test PCR is compared with the
amounts synthesized during PCRs with known
quantities of starting DNA.
• Quantification is carried out by real-time PCR:
two ways
➢ dye that gives a fluorescent signal with double-
stranded DNA BUT gives over-estimation because
sometimes the primers anneal to one another
A short oligonucleotide called a reporter probe, which
gives a fluorescent signal when it hybridizes with the
PCR product, can be used. Because the probe only
hybridizes with the PCR product, more accurate.
Quantitative PCR (qPCR):
 The first way to use fluorescent DNA intercalating
dyes. For example: SYBR Green I (an asymmetrical
cyanine dye used as a nucleic acid stain in molecular
biology) was used since it incorporates into double
stranded DNA.
As the amount of double stranded DNA increases
exponentially during the PCR reaction, the amount of
dye incorporation and emission also increases and is
measured.
An advantage to using SYBR Green is that since no special
probes are required, the cost is much cheaper. However,
SYBR Green will bind to any double-stranded DNA species
including nonspecifically amplified products leading to an
increase in background and false positives.
The second way of real-time PCR was performed using
hydrolysis or TaqMan probes. These probes specifically
hybridize to the region around a target allele internal to the
primer binding sites. The TaqMan probe is generally
labeled on each end with a fluorescent molecule, a reporter
dye, and a quencher. As long as the two are nearby, the
quencher prevents the reporter from fluorescing.

As the PCR cycle progresses, the exonuclease activity of


Taq polymerase degrades the probe and the fluorophore
becomes separated from the quencher allowing
fluorescence emission which can then be measured. The
increase in fluorescence is measured at every cycle and
directly correlates to the amount of PCR product formed.

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