PCR AND ITS TECHNIQUES

Presented by: Mobeen Ghazanfar

M. Phil (FIRST)

What is PCR?

It was invented in 1983 by Dr. Kary Mullis, for which he received the Nobel Prize in Chemistry in 1993.

It is a molecular technology aim to amplify a single or few copies of the DNA to thousands or millions of copies

PCR

It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.It is called “chain” because the products of the first reaction become substrates of the following one, and so on.

The “Reaction” Components

Target DNA - contains the sequence to be amplified

Pair of Primers - oligonucleotides that define the sequence to be amplified

dNTPs – deoxynucleotide triphosphates: DNA building blocks.

Thermostable DNA Polymerase - enzyme that catalyzes the reaction

Mg++ ions - cofactor of the enzyme

Buffer solution – maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme

Temperature Protocol

– Initial Melt: 94ºC for 2 minutes– Melt: 94ºC for 30 seconds– Anneal: 55ºC for 30 seconds– Extend: 72ºC for 1 minute– Final Extension: 72ºC for 6 minutes– Hold: 4ºC

30-35 cycles

Applications of PCR• Mutation screening• Classification of

organism• Bioinformatics• Genomic cloning• Gene expression studies

• Detection of pathogens• pre-natal diagnosis• DNA fingerprinting• Gene therapy's• Parental testing• identification of species

from fecal pellet

PCR TECHNIQUES

Multiplex PCR

• amplify several different DNA targets (genes)• Perform many separate PCR reactions all

together in one reaction• multiple primers and a temperature-

mediated DNA polymerase in a thermal cycler• Specific primers are used for different targets

because they attach to specific DNA sequence at similar temperature

application of multiplex PCR includes:

• pathogen identification• Mutation analysis• Gene deletion analysis• RNA detection • Forensic studies

HOT –START PCR• reduce non-specific amplification during the initial

set up stages of the PCR• heating the reaction components the melting

temperature (e.g., 95 C) before adding the polymerase• Specialized enzyme system inhibit the polymerase’s

activity at room temperature, either by the adding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high temperature activitation step

• Hot start PCR new hybrid polymerase that are inactive at ambient temperature

• instantly activated at elongation temperature 72 C

Nested PCR

• Reduce nonspecific amplification• To get specific amplification • two sets of primers are used,• allowing double process of amplification• increases the sensitivity to small amounts of

the target sequence• Forensic• Analysis sequence

REVERSE TRANSCRIPTASE PCR (RT-PCR)

Detect RNA expression• RNA sequence can be amplified by

using of mRNA • complementary DNA• reverse transcriptase (RT)• diagnosis of RNA viruses • study of gene expression in vitro due to

the obtained cDNA

Reverse transcriptase PCR

REAL TIME PCR

• quantitative PCR • quantifies the number of copies of

nucleic acid• calculates the amount of DNA, cDNA

and RNA• uses fluorescence detection dyes such as

Sybr Green

Fluorescent reporter probe method

MULTIPLEX LIGATIONDEPENDENT PROBE AMPLIFICATION (MLPA-PCR)

• amplifies small quantities of DNA • allow for multiple loci to be tested in a single

reaction• Multiple targets amplified with a single primer

pair • avoids the resolution limitation of multiplex PCR• reliable, and effective for rapid screening of

multiple loci for copy number changes

MLPA-PCR

• synthetic probes are used for anealing to their genomic target sequence

• ligated together at 54°C using thermostable DNA ligase

• PCR can screen at least 40 loci in one reaction with as little as 20 ng template DNA

• DNA ligase is exquisitely sensitive to DNA mismatches,

Applications

to obtain the target specificity and discrimination

detection of mutations  single nucleotide polymorphisms analysis of DNA methylationdetection of duplications and deletions in

human cancer

METHYLATION SPECIFIC PCR

• Determine the methylation status of DNA• (cancer) the genes which control cellular

division are present but switched off• this region contain methyl group to the

carbon-5 position of cytosine• Used to identify patterns of DNA

methylation at cytosie guanin (CpG)

MSP PCR• Target DNA is first treated with sodium

bisulphite, which converts unmethylated cytosine base to uracil, which is complementary to adenosine in PCR primers

Two amplification are carried out on the bishulphite treated DNA:

• One primer set aneal to DNA with cytosines (corresponding to methylated cytosine)

• Other set aneal to DNA with uracil (corresponding to unmethylated cytosine)

INVERSE PCR

•  primers oriented in the reverse direction of the usual orientation

• Known sequence at the centre flanked by unknown sequence

• target DNA is lightly cut into smaller fragments of several kilobases by restriction endonuclease digestion

• This technique involves the self ligation and DNA digestions to produce known sequence

Inverse PCR

Applications• to amplify and clone unknown DNA• Identification of genomic inserts

INTER SIMPLE SEQUENCE PCR

• amplify the regions between simple sequence repeats

• no prior genomic information is required • amplifies large number of DNA fragments per

reaction representing multiple loci from across the genome

ISSR-PCR

ISSP-PCR

• Applications • genetic identity, parentage, • clone and strain identification, • taxonomic studies of closely related species• gene mapping

Helicase dependent amplification PCR

• This is similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and anealing/ extension cycles

• DNA helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation

Colony PCR

The screening of bacterial (E.Coli) or yeast clones for correct ligation or plasmid products

Pick bacterial colony with autoclave toothpick Heat in a boiling water bath (100 C) 2 mints Centrifugation at high speed 2 mints Transfer supernant in new microfuge tube 1-2 ul of supernant as template in PCR reaction

In situ PCR (ISH)

• A polymerase chain reaction that actually takes place inside the cell on the slide. In situ PCR amplification can be preformed on fixed tissue or cells

Widely used in areas such as • Embryogenesis • Organogenesis • Infectious

diseases • Genetics

applications • Detection and diagnosis of viruses and infectious agents in

specific cell types within tissues• Detection and characterization of tumor cells within tissues• Detection and diagnosis of genetic mutations in inherited

diseases

• Detection of gene and gene expression in a tissue

• Main advantages are high specificity, fast assay with shorter turn around time and no need of radioactive chemicals

SINGLE CELL PCR

• To amplify and examine minute quantities of rare genetic material: limit of this exploration being the single cell

Applications in the field of prenatal diagnostics• Preimplantation genetic analysis and the use of fetal

cells enriched from the blood of pregnant women for the assessment of single gene Mendelian disorders

• use in basic scientist investigating immunological, neurological and developmental problems

Long PCR

PCR, which extended or longer than standard PCR, meaning over 5 kilobases (frequently over 10 kb)

Applications of Long PCR Long PCR is often used to clone larger genes or large segments of DNA 

PCR-Restriction Fragment Length Polymorphism (PCR-RFLP)

• in genome mapping and genetic disease analysis

• exploits variations in homologous DNA sequences

•  homologous DNA molecules from differing locations of restriction enzyme sites,

• broken into pieces (digested) by those restriction enzymes

• resulting fragments are separated according to their lengths by gel electrophoresis

Allele specific PCR• A diagnostic or cloning technique which is based on

single-nucleotide polymorphism (SNPs), single base difference in DNA

• It requires prior knowledge of a DNA sequence, including difference between alleles

• PCR amplication is much less efficient in the presence of a mismatch between template and primer

• So successful amplication with an SNP –specific primer signals presence of the specific SNP in a sequence

Asymmetric PCR

• This PCR amplifies one DNA strand in a double stranded DNA template

• It is used in sequencing and hybridization probing where the amplification of only one of the two complementary strands is required

• PCR is carried out as usual, but with a great excess of the primer for the strand targeted for amplification

Degenerate PCR• mixed PCR primers will be used.• That is “wobbles” are inserted into the primers

in case if the exact sequence of gene is not known,

• so that there will be more than one possibility for exact amplifications

• it has proven to be a powerful tool to find ‘new’ gene or gene families

• By aligning the sequences from a no. of related proteins the conserved and variables part can be determined