Download Application of PCR-technique in biological labs

Application of PCR-technique in
biological labs
Arsalaan naveed
Ayesha naeem
Bdar slam Saba naseem
Uzair hashmi
Applications of PCR in Diagnostics
PCR for pathogen detection
other methods for detection
1. Cultures and colony counting assay
•Oldest bacterial detection method
•Culturing methods are extensively time consuming
2. Immunology Based Methods :
•Antigen-antibody interactions
•Doesn't tell the pathogen load (serum-viral load)
PCR
A Nucleic acid amplification technology
“Widely used in pathogen detection”
1.
2.
3.
Basic steps
Isolation of DNA
Amplification of DNA
Quantification of DNA sequence (targeted
pathogen’s genetic material)
Priority over other detection methods
Today the detection methods have been replaced by
PCR due to
•Quick results (culturing takes even weeks)
•More specific (no false positive results, as a result of
contamination)
•Quantification of serum viral load , (pathogen titter
from sample)
PCR pathogen detection assays
Principle :•Amplifies target nucleic acid
sequence from microbes present
in the samples
This amplification of target
sequence is achieved by
•Custom designed probes
•Custom designed primers
Contd...
First degree of specificity :
Achieved by combination of amplification
primer sequence
Additional degree of specificity :
Achieved by the hybridization of the probe
To a
Region of nucleic acid sequence that identifies the
microbe of interest
Specificity and sensitivity
Microbe’s genome :Specificity of microorganism is due to a
target (specific) sequence in its genome.
This sequence encodes their virulent
agents.
How to distinguish microbe from its nearest
neighbour ?
The gene (or its portion) that contribute to a disease
phenotype of the pathogen:
distinguish a target microbe with its nearest neighbour
Distinction and detection
Target sequence needs to be identified
•Highly specific and unique for a
particular species (this target sequence
will be )
Specificity implies two properties
Exclusion
Inclusion
Assay will detect all strains Assay will not detect
of target species
neighbour species
Detection of HIV by IN situ PCR
HIV has the ability to infect different cell types and tissues
Principle :
HIV nucleic acid sequence is amplified using specific primer pairs.
•Amplification of target DNA
•Hybridization with 3’ end labelled oligonuecleotide
•Detection with anti-DIG-AP
Amplification of target DNA
Amplification of
HIV DNA was
carried
out on slides, using
primer pairs from
the gag
(SK38/39) region
(3), and performed
on a
thermo cycler.
Hybridization
Hyb. With DIG-labelled
oligonucleotide probe
Detection
Using polyclonal antibody
DIG label was detected by
Alkaline phosphate
conjugated polyclonal
antibody
HBV AND HCV DETECTION USING
PCR
• Detection of HCV RNA in patient specimens by
polymerase chain reaction (PCR) provides:
• Evidence Of Active HCV Infection
• Is Potentially Useful For Confirming The
Diagnosis And Monitoring The Antiviral
Response To Therapy.
TECHNIQUE USED
reverse transcriptase-PCR- (RT-PCR-)
based assays
Qualitative RT-PCR
HCV RNA is used as a matrix
• HCV reverse
RNA is used
as a matrix
transcriptase
used
here
synthesis of a single-stranded
complementary cDNA
DNA polymerase>The cDNA is
then amplified
multiple double-strandedDNA
copies.
Target Amplification Techniques
real-time PCR
competitive
PCR
Quantitative HCV
RNA detection
Quantification is achieved by:
AMPLIFICATION
INSIDE
THE REACTOR TUBE
TWO
TEMPLATES IN A
SINGLE
REACTION
TUBE
THE TARGET
THE INTERNAL
STANDARD
• Comparison of the final amounts of both templates
allows calculation of the initial amount of HCV RNA.
• The internal standard is an internal control RNA with
nearly the same sequence as the target RNAwith a
clearly defined initial concentration.
• The internal control is amplified by the
same primers as the HCV RNA.
• It has a specificity of almost 100%, independent of the
HCV genotype
Real-Time PCR Assay for Detection
and Quantification of Hepatitis B
Virus Genotypes A to G
• The detection and quantification of hepatitis
B virus (HBV) DNA play an important role in
diagnosing and monitoring HBV infection as
well as assessing therapeutic response
• The great variability among HBV genotypes
and the enormous range of clinical HBV DNA
levels present challenges for PCR-based
amplification techniques
PCR assay designed to provide accurate
quantification of DNA from all eight HBV
genotypes in patient plasma specimens.
1
2
3
• If the target of interest is present during PCR, the
probe specifically anneals between the forward
and reverse primers
• The 5′-3′ exonuclease activity of Taq polymerase
cleaves the probe between the reporter and the
quenche
• This results in an increase in fluorescence of the
reporter that is proportional to the amount of
product accumulated
• Following amplification, real-time data acquisition
and analysis are performed
RT-PCR AMPLIFICATION PLOT
Tuberculosis
•Limitation in culturing
•Species are slow growing
Needs 6-8 weeks for growing
•Species can be contaminated while growing
RESULT :Specificity is lost due to contamination
And can also yield false positive results
PCR based TB diagnostic test
•Sputum
•Sample processing
•DNA extraction
• AMPLIFICATION of MTB DNA
•123 bp DNA fragment is amplified using specific
primers
• amplified products were analysed by
electrophoresis
•123bp specific band detected by gel doc TRANS
illuminator
PCR based dengue detection
Identification of dengue virus
2-step PCR reaction
1.Reverse transcription
2.Amplification using universal
dengue primers
Reverse transcription
RNA strand is reverse transcribed into DNA using reverse
transcriptase
cDNA is amplified using RT-PCR
Universal dengue primers are used
Targeting a specific region of viral genome
•PCR products are separated on gel electrophoresis
Different size bands are seen
•They are compared with a standard marker
•Tells the relative molecular mass of nucleic acids
dengue serotypes are identified by the types of bands
RT-PCR is one step assay system
Primers and probes
Specific for each genotype
Detection of pathogens in real time without using
the electrophoresis
Florescent probes are used
As singleplex
• Detecting one
serotype at a
time
As
multiplex
• All four
serotypes
• From
single
sample
Multiplex has
advantage
• All 4 serotypes
at a time
• No
contamination
Nested
PCR
M.RT -PCR
More
sensitive
Les sensitive
?
Determine
viral load in a
sample
PCR in Prostate Cancer detection
Methylation specific PCR (msp) technology
DNA is methylated only at certain cytosine located 5' to a
guanosine.
This occurs especially in GC-rich regions, known as CpG
islands
Obj :- methylation state of sequence
How to achieve this ?
Chemical modification in the cytosine residues in DNA
Sod. Bi sulphite will
convert all the
unmethylated Csequence residues into
uracil
Methylated cytosine will
remain same
Different DNA sequence will be formed for methylated and unmethylated DNA .
PCR primers will distinguish
Primers will anneal to
the unchanged
cytosines (that are
methylated in the
gene.
Primer will anneal with
altered cystosine(uracilthat were un-methylated)
Comparison will reveal the methylation state of DNA
Primer set with altered sequence gives a
product ?
Indicated cytosine were un-methylated
Primer set with unchanged sequence
gives a product?
Cytosine was methylated and protection from alteration
Prostate cancer : aik tak’nikee kharabi
Genetic alteration in prostate carcinoma
Hyper-methylation of GSTP1 promotor
GSTP1 : maker for detection and
molecular staging of p.c
Function of GSTP1 :
•Involved in
intracelleular
detoxification
reaction
•Candidate tumor
suppressor gene in
Pros.Cncr
•Hyper methylation
results in loss of gene
expression
In Chronic Myelogenous Leukemia
Cancer of WBC
Increased and uncontrolled growth of myeloid
cells in bone marrow.
Genetic abnormality
Chromosomal translocation ,
formation of phaliadelpia chromosome
BCR gene in 22 , fused with abl gene in 9
p210
p210
Add phosphate group to
the tyrosine :tyrosine
kinase
Activates protien cascade that control cell cycle
Inhibits DNA repair .
RT-pcr comes into action
RNA is extracted
Subjected to RT-PCR
3 –types of primary transcripts of (bcr/abl gene)are
amplified
•B2a2
•B3a2
•E1a2
If there is no amplification of BCR-abl fusion
mRNA result will be reported as negative
RESEARCH
APPLICATIONS OF PCR
AYESHA NAEEM
GENE CLONING
Major application of PCR
PCR can produce large quantities of DNA that
can be readily cloned and used to study the
functions and behavior of genes in living
systems.
THE PCR STEPS
Denaturation
Annealing (60-70C)
Elongation (72C)
PCR-mediated cloning is a family of methods
rather than a single technique.
 TA cloning
Blunt-end cloning
TA CLONING
uses Taq polymerase and
Tth DNA polymerase
that preferentially add
adenine (A) to the 3' ends
of the PCR products.
These products are cloned into a vector
containing complementary overhangs of the
base thymidine (T).
BLUNT-END CLONING
uses DNA polymerases that possess
proofreading activity, such as Pwo DNA
polymerase.
They remove mispaired nucleotides from
the ends of double-stranded DNA and
generate blunt-end PCR products.
SELECTIVE DNA ISOLATION
PCR allows isolation of DNA fragments from
genomic DNA by selective amplification of a
specific region of DNA.
Thus, PCR provides high amounts of pure DNA
to be used as probes for Southern or Northern
hybridization and as primers for DNA cloning.
GENE EXPRESSION STUDIES
Reverse transcription quantitative polymerase
chain reaction (RT-PCR followed by qPCR) is
the gold-standard technique for measuring
gene expression.
sensitivity
broad dynamic range
lower-cost of instrumentation and reagents
mRNA quantification
qRT-PCR is a highly sensitive technique in which a
very low copy number of RNA molecules can be
detected
i. RT-PCR first generates a DNA template from the
mRNA by reverse transcription, called cDNA.
ii. cDNA template is used for qPCR where the
change in fluorescence of a probe changes as
the DNA amplification progresses.
iii. With a carefully constructed standard curve,
qPCR can produce an absolute measurement of
the number of copies of mRNA, in units of
copies per nanolitre of homogenized tissue .
Gene Mapping
RT-PCR is widely used to identify the sequence of
an RNA transcript, including transcription start
and termination sites.
If the DNA sequence of a gene is known, RT-PCR
can be used to map the location of exons and
introns in the gene.
The 5' end of a gene (corresponding to the
transcription start site) is typically identified by
RACE-PCR (Rapid Amplification of cDNA Ends).
Expression Of Eukaryotic Genes In
Prokaryotes
RT-PCR is very useful in the insertion of eukaryotic
genes into prokaryotes.
Most eukaryotic genes contain introns in the
genome but not in the mature mRNA, the cDNA
generated from a RT-PCR reaction is the DNA
sequence which is directly translated into
protein after transcription.
When these genes are expressed in prokaryotic
cells for protein production or purification, the
RNA produced from transcription need not
undergo splicing as it contains only exons.
Alteratins In Gene Expression
In research, real-time PCR is used in determining
how the genetic expression of a particular
gene changes over time, such as
in the response of tissue and cell cultures to
administration of a pharmacological agent
progression of cell differentiation
 in response to changes in environmental
conditions.
GENOTYPING
The process of determining differences in the
genetic make-up (genotype) of an individual,
by examining the individual's DNA sequence
and comparing it to another individual's
sequence or a reference sequence.
It reveals the alleles an individual has inherited
from their parents.
PCR IN GENOTYPING
Genotyping by PCR is used for screening alleles
based on gene structure.
An effective and efficient method for
detecting gene insertions, deletions, or
rearrangements in natural or artificial gene
constructs.
Alleles in any organism are detected by
identifying unique nucleotide elements in the
target gene of DNA (or RNA via cDNA) at the
PCR amplification stage, in the PCR product
Rapid
Reliable
Low cost and feasibility
High sensitivity
High resolution
of PCR make it is highly practical and valuable in
studies.
SNP GENOTYPING
The measurement of genetic variations of single
nucleotide polymorphisms (SNPs) between
members of a species i.e.
a base pair substitution at a specific locus within
a DNA sequence.
SNP genotyping is used to identify heritable
differences among individuals within a
population.
Tetra-primer ARMS-PCR
 Tetra-primer ARMS-PCR employs two primer
pairs to amplify the two different alleles of SNP.
 The primers are designed such that the two
primer pairs overlap at a SNP location but each
match perfectly to only one of the possible SNPs.
 If a given allele is present in the PCR , only the
primer pair specific to that allele will produce a
product.
 The two primer pairs are also designed such that
their PCR products are of a different length, to
easily distinguish bands by gel electrophoresis.
SNP Genotyping
 in studying genetic determinants of complex
diseases like sickle cell anaemia.
selective breeding is accelerated by allowing
traits to be identified and selected prior to
growing the organism to maturity. Homozygous
and hemizygous transgenic mice can be
distinguished using Quantitative PCR (qPCR).
The use of SNPs is being extended in the HapMap
project, which aims to provide the minimal set of
SNPs needed to genotype the human genome.
Application of PCR in Gene Therapy,
Human Genome Project & Drug
Discovery
Arsalaan Naveed
GENE THERAPY
• Gene therapy involves
diagnosing, treating and curing
diseases on molecular level
• What kind of tools do the
scientists use for such an
intricate methodology?
• How do they go about working
with ease?
• The answer lies in the vast
variety of specialized tools for
the trade of gene therapy
Why PCR?
• PCR is a molecular copying machine , which can
amplify DNA quickly and efficiently.
• Sample is first heated to denature the DNA
molecule into two separate strands.
• Taq polymerase is used to synthesize two new
strands complementary to the two templates.
• Each new strand contains one old and one new
strand.
• These synthesized strands can be used to create
further new copies.
Mechanism of PCR
Polymerase
Chain
Reaction
(or PCR)
helps
scientists
in their
study of
DNA
Contd. .
• Thermocycler is used to automatically
denature and synthesize DNA molecules.
• Millions of copies of DNA can be generated in
a relative short time.
• Using PCR, scientists can replicate DNA quickly
in order to test developed gene therapies and
the effect of a gene therapy on a DNA
molecule.
The Basics of the Procedure
• DNA is extracted.
• A chemical process called polymerase chain reaction
(PCR) uses enzymes to amplify the amount of DNA.
• Sections of DNA where repeats are present
are cut in order to determine the number of repeats.
• The fragments are put on an electric field that sorts them
by size (gel electrophoresis).
• The fragments are then placed onto a nylon membrane
where they are treated with radioactive probes.
Contd. . .
• The probe sticks to some DNA fragments but not
to others, due to complimentary base pairing.
• A piece of X-ray film is put on the top and a spot
is produced on the film where the probe sticks.
• Using a ruler, scientists measure the position of
the spots on the film and produce a set of numb
ers.
• The odds of two individuals having the same
pattern are between 1,000 to 1-to trillions- 1
Restriction Endonucleases
• Desirable genome lengths using restriction
endonucleases, which cut the DNA at specific
points.
• The particular gene is isolated in the form of
bands produced in gel electrophoresis
technique.
• The desired band can be amplified along with
the required gene.
Vectors in Gene therapy
• Vectors are the entities used to transfer genes
from one organism to another.
• Gene therapy requires the treatment &
manipulation at DNA or molecular level.
• Vectors are usually around the size of DNA
being used or are specially designed.
Types of vectors
• Viral Vectors
Retroviruses, adenoviruses ,adeno-associated
viruses, herpes simplex viruses etc
• Non-Viral Vectors
liposomes, naked DNA, plasmids, BAC, YAC
Viral Vectors
A type of viral
vector:
Adenovirus.
Using
adenoviruses,
desired DNA can
be quickly
moved into the
cell. The virus is
already
designed by
nature itself for
efficient entry
into the cell.
Non-Viral Vectors
Using plasmids
to transfer
DNA from one
organism to
another.
Plasmids are
considered
best in nature
for this
purpose
PCR based Gene Therapy
• It is being carried out for the production of
various gene products used for the treatment
a number of genetic and developmental
disease e.g. colorblindness, diabetes,
emphysema, cystic fibrosis, cancer, somatic
cell and germ-line therapy.
Examples
Product
Use
Host Organism
Insulin
human hormone used to
treat diabetes
bacteria /yeast
Factor VIII
human blood clotting
factor, used to treat
hemophiliacs
bacteria
AAT
enzyme used to treat cystic sheep
fibrosis and emphysema
Rennin
enzyme used in
manufacture of cheese
bacteria /yeast
Limitations in Effective Gene-Therapy
•
•
•
•
Short-lived nature of gene therapy
Immune response
Problems with viral vectors
Multigene disorders e.g. heart attack, high
blood pressure, Alzheimer’s disease, arthritis.
HUMAN GENOME PROJECT
What is Genome?
• It is the full collection of genetic material
(DNA) of an organism.
• It is more than the genes (which are about 3 %
of the human genome)
• In humans there are 3,000,000,000 base pairs
of DNA.
NEED for a GENOME PROJECT
• Since there are only four nucleotides which
are strung together without any punctuation.
• There are no signals to tell us where the gene
starts and where it ends.
• How to make sense of such an un-organized
information?
How it started?
• HGP was planned in 1988, started in 1990 and
was expected to be completed in 15 years.
• Objections:
i) Fear that funding will be diverted from
others areas of research.
ii) Worthless to sequence a complete genome
containing major portion as JUNK DNA.
Modification in the GOAL
• Focus moved from large scale sequencing to
mapping the genome, to hasten the search for
the disease gene.
• Simultaneously determining the nucleotide
sequence of the genomes of different
organisms to provide a comparison and point
o reference for the human genome.
AIMS of the Project
•
•
•
•
To create a genetic map of the genome.
To create a physical map of the genome.
To create a set of overlapping clones.
To create faster and cheaper methods of
sequences.
• Create software and databases that can deal with
the data.
• Sequencing
• To start annotation-gene finding and placement
on maps.
PCR -BREAKTHROUGH
• Improvement in sequencing was conferred by:
i) Cycle sequencing
ii) Automated sequencers
iii) Flourescent dyes
iv) PCR
USE OF PCR IN HGP
• Researchers select the desired genes and
heat-separate the DNA strands containing that
genes.
• Primers bind to complementary DNA
sequence ends and initiate synthesis.
• Nucleotides fill in the middle to form a
complete second strand.
• Multiple copies of desired segments are
obtained.
PCR Action
Detection
• Fluorescent in-situ hybridization
(FISH) is then used to detect the
desired genes in DNA segments.
• By using different colors for
fluorescent binding, one can
paint the genes on the
chromosomes, and can
ascertain their faulty location
• Genes that are
misplaced/missing cause
genetic diseases
Contd. .
Maps & Markers:• RFLP
Restriction Fragment
Length
Polymorphism(RFLP)
• Microsatellite e.g
CACACA/CAGCAGCAG
• VNTR (Variable
Number Tandem
Repeat)
• STS (Sequence
Tagged Site)
PCR IN DRUG DISCOVERY
Real time PCR
• Widely used for drug research and
development
Applications include:i) Genotyping
ii) Vaccine studies
iii) Discovery and validation of bio-markers
Results:Increased efficacy and less adverse effects
Right bio-marker for the right drug
• Identification of biomarker leads to :i) Identification of disease type
ii) Measurement of disease progress
iii) Increase the success rate of the drug
iv) Assist regulatory approval of clinical trials
v) Excluding non-responsive population to the
drug
Benefits and Procedure
• Real time experiments are based on relative quantification
• Real time PCR is easy and reliable to achieve normalization
Methods
i) Two genes are chosen i.e. target and the reference gene
(housekeeping gene).
ii) Both are amplified, and the expression of the target gene
is normalized to that of the ref. gene.
iii) Normalization provides an internal control that would
otherwise lead to inaccurate quantification e.g. variation
in input sample, sample degradation, presence of
inhibitors, difference in sample handling.
Example
• Target gene :- Myogenin
• Reference gene:- GAPDH
Both of them were amplified in the same
reaction mixture. Myogenin expression was
examined in untreated as well as treated cells.
In 5 independent experiment Myogenin
expression were detected with high
reproducibility.
APPLICATIONS OF PCR
BY
SABA NASIM AWAN
CONTENTS
• Applications of PCR in DNA fingerprinting
Criminal Cases
Medical Cases
• Applications of PCR in DNA footprinting
DNA-Protein interactions
• In Forensics the field of DNA fingerprinting
relies on PCR.
• Significance of using PCR is that it employs
DNA for detection which is present in all body
cells.
• PCR determines the unique DNA “fingerprint”
of victims or suspects.
DNA FINGERPRINTING
(DNA PROFILING)
A technique used by scientists to distinguish
between individuals of the same species using
only samples of their DNA
INVENTER
• The process of DNA
fingerprinting was
invented by Alec
Jeffreys at the
University of
Leicester in 1985.
• He was knighted in
1994.
Biological materials used for DNA
profiling
•
•
•
•
•
Blood
Hair
Saliva
Semen
Body tissue cells
Stages of DNA Profiling
Stage 1:
Cells are broken down
to release DNA
If only a small amount of
DNA is available it can be
amplified using the
polymerase chain reaction
(PCR)
Stages of DNA Profiling
Stage 2:
• The DNA is cut into fragments using restriction enzymes.
• Each restriction enzyme cuts DNA at a specific base sequence.
Stages of DNA Profiling
• The sections of DNA that are cut out are called
restriction fragments.
• This yields thousands of restriction fragments
of all different sizes because the base
sequences being cut may be far apart (long
fragment) or close together (short fragment).
Stages of DNA Profiling
Stage 3:
• Fragments are separated
on the basis of size
using a process called
gel electrophoresis.
• DNA fragments are
injected into wells and
an electric current is
applied along the gel.
Stages of DNA Profiling
• DNA is negatively
charged so it is attracted
to the positive end of
the gel.
• The shorter DNA
fragments move faster
than the longer
fragments.
• DNA is separated on
basis of size.
Stages of DNA Profiling
• A radioactive material is
added which combines
with the DNA fragments
to produce a fluorescent
image.
• A photographic copy of
the DNA bands is
obtained.
Stages of DNA Profiling
Stage 4:
• The pattern of fragment distribution is then
analysed.
Uses of DNA Profiling
• DNA profiling is
used to solve crimes
and medical
problems
Crime
• Forensic science is the use of scientific
knowledge in legal situations.
• The DNA profile of each individual is highly
specific.
• The chances of two people having exactly the
same DNA profile is 30,000 million to 1
(except for identical twins).
DNA Profiling can solve crimes
• The pattern of the DNA profile is compared with
those of the victim and the suspect.
• If the profile matches the suspect it provides strong
evidence that the suspect was present at the crime
scene (NB:it does not prove they committed the
crime).
• If the profile doesn’t match the suspect then that
suspect may be eliminated from the enquiry.
CRIMINAL CASES
• Colin Pitchfork was the
first criminal caught
based on DNA
fingerprinting evidence.
• He was arrested in 1986
for the rape and murder
of two girls and was
sentenced in 1988.
CRIMINAL CASES
• O.J. Simpson was
cleared of a double
murder charge in 1994
which relied heavily on
DNA evidence.
• This case highlighted
lab difficulties.
Solving Medical Problems
DNA profiles can be used to determine whether a
particular person is the parent of a child.
A childs paternity (father) and maternity(mother) can be
determined.
This information can be used in
• Paternity suits
• Inheritance cases
• Immigration cases
PCR IN FORENSICS
Hyper variable microsatellite sequence (VNTR)
Runs of short repeated DNA sequences
Inheritance from parents
PCR IN FORENSICS
PCR IN FORENSICS
1)
2)
3)
4)
PCR AMPLIFICATION
2 primers are used for each VNTR
Primers bracket the locus of VNTR
For each VNTR 2 DNA bands are generated
After electrophoresis, bands are positioned
according to their exact no of repeats.
PCR IN FORENSICS
MITOCHONDRIAL DNA ANALYSIS
• For degraded or old biological material that
lacks nuclei e.g., hairshafts, bones and teeth etc
• For maternal relationships
MECHANISM
• Hyper variable Control Regions (HVR1 or
HVR2) are used for detection of maternal lineage.
CASE: Anna Anderson was not the Russian
princess who claimed to be Anastasia Romanov.
Y-Chromosomal DNA paternity
Y-STR analysis can help
in the identification of
paternally related males.
In 2002 Elizabeth Hurley
used DNA profiling to
prove that Steve Bing
was the father of her
child Damien
PCR IN DNA FOOTPRINTING
•
This technique is used to assess whether a given protein binds to a region of interest
within a DNA molecule.
•
Polymerase chain reaction (PCR) amplifies and labels region of interest that
contains a potential protein-binding site.
•
Protein of interest is added to a portion of the labeled template DNA
•
A cleavage agent, with sequence independent cleavage, is added to both portions of
DNA template. It cuts each DNA molecule in only one location.
•
Both samples are run side by side on a polyacrylamide gel electrophoresis. The
portion of DNA template without protein will be cut at random locations, and thus
when it is run on a gel, will produce a ladder-like distribution. The DNA template
with the protein will result in ladder distribution with a break in it, the "footprint",
where the DNA has been protected from the cleavage agent.
PCR IN DNA FOOT PRINTING
Applications of PCR in :
 Mutagenesis (site directed mutagenesis)
 Prenatal Diagnosis
 Mutation Detection
BADAR UL SLAM
Site-directed Mutagenesis
using PCR
• Used for introducing mutations at the desired
place in a DNA sequence by altering the
sequences of primers
• Since mutations are introduced only through
primers, mutations are limited to the ends of the
gene sequence.
• Allows mutations to be introduced at any place of
interest in the gene
Site-directed Mutagenesis
using PCR
Design two sets of primers,
one set containing the
desired mutation
Extend each primer with DNA
polymerase
Denature and re-anneal the
DNA strands to produce
heteroduplexes
Only one heteroduplex can
be extended from 3’ to 5’
PCR in prenatal diagnosis
• Since 1987, PCR has had a major impact on prenatal
diagnosis of single gene disorders.
• QF PCR is used in laboratory of human genetics to
detect the common numeral chromosomal
abnormalities of chromosomes 21, 18, 13, X and Y.
trisomies 13, 18 and 21 are detected with about 99%
accuracy, usually within 48-72 hours and at a very
low cost.
• Improved speed, accuracy and technical flexibility
over previous methods.
PCR in prenatal diagnosis
• For prenatal diagnosis, PCR used to amplify DNA from
fetal cells obtained from amniotic fluid.
• Single base changes then detected by one or more of
following:
-dot blot (spot hybridization) with oligonucleotides
specific for known mutation.
-restriction enzyme analysis (RFLP).
-direct sequencing of DNA.
• Important to be certain of result so combination of two
methods provides confirmation.
PCR in prenatal diagnosis
• Many other conditions can be detected with same approach,
including:
-Tay-Sachs disease, phenylketonurea, cystic fibrosis,
hemophilia, Huntingdon's disease, Duchenne muscular
dystrophy (DMD).
• The PCR product in all these cases is examined using a
labelled probe, to suggest whether or not mutant sequence
causing the disease is found or not
• In some cases RFLP pattern of PCR products in healthy and
defective fetus differ, thus enabling prenatal diagnosis
• In still other cases PCR product may be sequenced to reveal
the difference
PCR for mutation detection
• Two types of PCRs used:
Real Time PCR (RT-PCR)
Allele specific PCR with Blocking reagent
(ASB-PCR)
PCR for mutation detection
• RT-PCR, a hybridization-based method, has
become widely used for mutation detection
• Different probe systems can be used:
 hybridization probes,
 hydrolysis probes,
 molecular beacons
 scorpion primers
PCR for mutation detection
• ASB-PCR can be used for detection of germ
line or somatic mutations in either DNA or
RNA extracted from any type of tissue
• A set of reagents developed enabling sensitive
and selective detection of single point
substitutions, insertions, or deletions against a
background of wild-type allele in thousandfold or greater excess.
Some Examples of Mutation
Detection by PCR
1. Detection of Fragile X CGG Expansion
premutations by PCR
2. Detection of Huntingtin Gene
Mutations by PCR
3. Detection of Mitochondrial Point
Mutation by PCR-RFLP
Fragile X syndrome and premutation
detection by PCR
• Fragile X syndrome (FXS), is a genetic
syndrome that is the most common inherited
cause of intellectual disability
• The syndrome is associated with the
expansion of a single trinucleotide gene
sequence (CGG) on the X-chromosome, and
results in a failure to express the protein
which is required for normal neural functions
Detection of Fragile X CGG Expansion
premutations by PCR
PCR
50–90
(pre-mutation)
20–40
(normal)
Pre-mutations can be
detected by PCR
Detection of Huntingtin Gene Mutations
by PCR
• The Huntingtin gene, is the IT15 ("interesting
transcript 15") gene codes for the huntingtin
protein(350 kDa)
• In its wild-type (normal) form, it contains 6-35
glutamine residues
• In mutated individuals, it contains greater than 36
glutamine residues
• The exact function of this protein is not known,
but in cells it plays an important role in signalling,
transporting materials, binding proteins and
protecting against apoptosis.
Detection of Huntingtin Gene Mutations by
PCR
Labeled PCR
primer
Huntingtin
80–170 bp
10–29 repeats
(normal)
Autoradiogram of polyacrylamide gel
>40 repeats
Huntington
Disease
Detection of Mitochondrial Point Mutation by PCRRFLP
U = Uncut, no MspI
C = Cut, with MspI
MspI U
C
U C U C
551 bp
The presence of
the mutation
creates an MspI
restriction
enzyme site in the
amplicon.
345 bp
206 bp
Agarose gel
Mutation
present
THE END