Application of PCR-technique in biological labs

Arsalaan naveed Ayesha naeem Bdar slam Saba naseem Uzair hashmi

Applications of PCR in Diagnostics

Uzair hashmi

PCR for pathogen detection

other methods for detection1. Cultures and colony counting assay •Oldest bacterial detection method•Culturing methods are extensively time consuming2. 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”

Basic steps 1. Isolation of DNA 2. Amplification of DNA3. 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Today the detection methods have been replaced by PCR due to •Quick results (culturing takes even weeks)•More specific (no false positive results, as a result of contamination)•Quantification of serum viral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

Inclusion Exclusion

Assay will detect all strains of target species

Assay will not detect 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 carriedout on slides, using primer pairs from the gag(SK38/39) region (3), and performed on athermo 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 RNA is used as a matrix

reverse transcriptase used here

synthesis of a single-stranded complementary cDNA

DNA polymerase>The cDNA is then amplified

multiple double-strandedDNA copies.

Target Amplification Techniques

Quantitative HCV RNA detection

competitive PCR

real-time PCR

Quantification is achieved by:

THE INTERNAL STANDARDTHE TARGET

THE REACTOR TUBEAMPLIFICATION INSIDE

TWO TEMPLATES IN A

SINGLE REACTION

TUBE

• Comparison of the final amounts of both templatesallows 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 thesame 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.

•If the target of interest is present during PCR, the probe specifically anneals between the forward and reverse primers

1

•The 5 -3 exonuclease activity of ′ ′ Taq polymerase cleaves the probe between the reporter and the quenche2

•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

3

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 contaminationAnd 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 transcriptionRNA strand is reverse transcribed into DNA using reverse transcriptasecDNA is amplified using RT-PCRUniversal 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

Florescent probes are used

Detection of pathogens in real time without using the electrophoresis

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

More sensitive

?

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 C-sequence residues into uracil

Methylated cytosine will remain same

Different DNA sequence will be formed for methylated and un-methylated DNA .

PCR primers will distinguish

Primers will anneal to the unchanged cytosines (that are methylated in the gene.

Primer will anneal with altered cystosine(uracil- that 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

Function of GSTP1 :•Involved in intracelleular detoxification reaction•Candidate tumor suppressor gene in Pros.Cncr•Hyper methylation results in loss of gene expression

GSTP1 : maker for detection and molecular staging of p.c

In Chronic Myelogenous Leukemia

Cancer of WBCIncreased 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

p210Add 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-PCR3 –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 CLONINGMajor 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 polymerasethat preferentially add adenine (A) to the 3' ends of the PCR products. These products are cloned into a vectorcontaining complementary overhangs of thebase 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 rangelower-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.

GENOTYPINGThe 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 feasibilityHigh sensitivity High resolution of PCR make it is highly practical and valuable in

studies.

SNP GENOTYPINGThe 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 numbers.

• 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 OrganismInsulin human hormone used to

treat diabetesbacteria /yeast

Factor VIII human blood clotting factor, used to treat hemophiliacs

bacteria

AAT enzyme used to treat cystic fibrosis and emphysema

sheep

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 sequencingii) Automated sequencersiii) Flourescent dyesiv) 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:-• RFLPRestriction 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) Genotypingii) Vaccine studiesiii) Discovery and validation of bio-markersResults:-Increased efficacy and less adverse effects

Right bio-marker for the right drug

• Identification of biomarker leads to :-i) Identification of disease typeii) Measurement of disease progressiii) Increase the success rate of the drugiv) Assist regulatory approval of clinical trialsv) 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 normalizationMethods 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

PCR AMPLIFICATION

1) 2 primers are used for each VNTR

2) Primers bracket the locus of VNTR

3) For each VNTR 2 DNA bands are generated

4) 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

Mutagenesis (site directed mutagenesis) Prenatal Diagnosis Mutation Detection

BADAR UL SLAM

Applications of PCR in :

Site-directed Mutagenesisusing 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 Mutagenesisusing 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.

• 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 in prenatal diagnosis

• Two types of PCRs used:

Real Time PCR (RT-PCR)

Allele specific PCR with Blocking reagent (ASB-PCR)

PCR for mutation detection

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

• 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 thousand-fold or greater excess.

PCR for mutation detection

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 (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

Fragile X syndrome and premutation detection by PCR

Pre-mutations can be detected by PCR

PCR

50–90(pre-mutation)

20–40(normal)

Detection of Fragile X CGG Expansion premutations 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

10–29 repeats(normal)

>40 repeatsHuntingtonDisease

Huntingtin

80–170 bp

Labeled PCR primer

Autoradiogram of polyacrylamide gel

Detection of Huntingtin Gene Mutations by PCR

551 bp206 bp345 bp

MspI U C U C U C

Agarose gel

U = Uncut, no MspIC = Cut, with MspI

Mutationpresent

Detection of Mitochondrial Point Mutation by PCR-RFLP

The presence of the mutationcreates an MspIrestrictionenzyme site in the amplicon.

Thankyou

THE END