Polymerase chain reaction

PCR

History 1971- K. Kleppe, E. Ohtsuka, R. Kleppe, I. Molineux∥, H.G.

Khorana, ‘Studies on polynucleotides: XCVI. Repair replication of short synthetic DNA's as catalyzed by DNA polymerases’.

1984- mullis presented the concept of PCR at cetus corporation. 1985- nature publications and Science rejected mullis paper for

publication. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich

HA, Arnheim N, ‘Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia’. Science. 1985 Dec 20;230(4732):1350-4. US Patent 4,683,202.

Mullis, K. B. & Faloona F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Meth. Enzymol. 155, 335–350 (1987)

First PCR device Mr. Cycle:- first semi automated PCR

device from PerkinElmer Cetus Instruments (PECI) (1985) collaboration.

First polymerase from E.coli:- Manual addition of fresh enzyme after every cycle of denaturation.

Used tubes to heat and cool water

First PCR deviceMr. Cycle

History Gelfand of cetus isolated taq polymerase

from thermus acqaticus.

Peltier heating and cooling devices were used- no manual addition of reagents (Thermostable Poly).

1989- Patent received by cetus for Taq polymerase.

HistorySon-of-son-of-Cycle Baby Blue

First device with Peltier heating and cooling system

First integrated peltier-based thermal cycler.

DNA thermal cycler 480

First commercial PCR device launched by PECI

The PCR business 1989- agreement of cetus with roche roche diagnostic systems opened roche diagnostic research

with cetus. 1989-91- 20 licences were made by cetus with commercial

reference labs to perform PCR based diagnostics. 1990- HLA DQ alpha forensic kit:- only product cetus handled

full rights. 1991- roche and perkin elmer announced that roche holds the

ownership of PCR, paying 300 million dollars to cetus. Peci dissolved and perkin elmer develops instruments and

distributes roche pcr reagents. Roche opened roche molecular systems (RMS). ROCHE and

perkin elmer made more than 400 licences with other firms to manufacture PCR reagents and instuments.

Components & compositions

DNA template DNA Polymerase Buffer Mg++ ions Primers Nucleotides Water

TEMPLATE The whole genomic DNA, plasmid DNA of bacteria

or viral DNA/RNA ( human genome-3,200 Mb). Our sequence/gene of interest resides. Optimal concentration- 0.1-1ug/ml. Theoretically it is possible to get several copies of

amplicons from a single molecule of DNA. The result of PCR depends on the quality of

template DNA.- polymerase inhibitors, proteases, DNAses, etc.

The choice of extraction decides the purity of the starting material.

TEMPLATE DNA quantification:-

UV spectrophotometry;-Principle- beer lamberts law

DNA exibits maximum absorbance of UV at 260 nm. At 260nm, an absorbance of 1 corresponds to 50ug/ml of DNA. Purity assesment; the ratio of 260/280 was used. A value of

~1.8 for DNA and ~2 for rna is of PCR grade.Nanodrop; - Technical advancement of UV spectrophotometry- Flourscent based analysers also avail- Autoanalyse the quality & quantity of DNA. Requires samples in

microvolumes. Storage; -20 C. frequent thawing and freezing to be avoided to

prevent degradation.

Nanodrop

DNA polymerase Concentration; 0.5 to 3U Higher concentrations required if the

template contains inhibitors. Taq- Thermus aquaticus Pfu-Pyrococcus furiosus ( 3'->5'

exonuclease activity) recombinant DNA polymerase; eg;

platinum Pfu- high fiedility

Buffer Provides suitable environment for the

enzyme activity. Contains, - mgcl2

-kcl – k+ stabilizes primer annealing-tris-hcl

Concentration depends on the amount of mgcl2 present.

Usually 10% i.e 5ul per 50ul reaction

MG2+ Co- factor for polymerase Act as catalyst Facilitates the formation of phosphodiester

bonds between nucleotides Stabilize primer annealing by shielding the

negative electrostatic repulsion between the primer and template.

Optimum concentration- 1-4mM Reaction buffers are supplemented along

with mgcl2

dNTPS dATP dTTP dCTP dGTP Optimum concentration- 0.2mM Decreased concentration will result in low

quantity of the end product Increasing the concentration does not

result in increased quantity of finalproduct

Types Allele-specific PCR: a diagnostic or cloning technique based

on single-nucleotide variations (SNVs) (single-base differences in a patient). It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends containsthe SNV (base pair buffer around SNV usually incorporated

Assembly PCR or Polymerase Cycling Assembly (PCA): PCR on a pool of long oligonucleotides with short overlapping segments. overlapping segments determine the order of the PCR fragments, thereby selectively producing the final long DNA product. Oder of genes.

Asymmetric PCR: preferentially amplifies one DNA strand in a double-stranded DNA template. It is used in sequencing and hybridization probing where amplification of only one of the two complementary strands is required

Types Digital PCR (dPCR): measure the

quantity of a target DNA sequence in a DNA sample. The DNA sample is highly diluted.some of them do not receive a single molecule of the target DNA. The target DNA concentration is calculated.

Helicase-dependent amplification: uses a constant temperature. DNA helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.

Types Hot start PCR: a reduces non-specific

amplification. heating the reaction components to the denaturation temperature (e.g., 95 °C) before adding the polymerase.

Intersequence-specific PCR (ISSR): a PCR method for DNA fingerprinting that amplifies regions between simple sequence repeats to produce a unique fingerprint of amplified fragment lengths.

Types Inverse PCR: identify the flanking

sequences around genomic inserts. series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown sequence.

Ligation-mediated PCR: uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers.

Inverse

Ligation-mediated PCR

Types Miniprimer PCR: uses a thermostable

polymerase (S-Tbr) that can extend from short primers ("smalligos") as short as 9 or 10 nucleotides. This method permits PCR targeting to smaller primer binding regions, and is used to amplify conserved DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene.

Multiplex-PCR: consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences.

Types Nested PCR: increases the specificity of DNA amplification,

by reducing background due to non-specific amplification of DNA.

Two sets of primers are used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction.

Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.

Nested

Types Reverse Transcription PCR (RT-PCR): for

amplifying DNA from RNA. Reverse transcriptase reverse transcribes RNA into cDNA, which is then amplified by PCR.

RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites.

If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of exons and introns in the gene.

Reverse Transcription PCR (RT-PCR)

QRT- PCR Based on the detection and quantitation of

a fluorescent reporter

In stead of measuring the endpoint we focus on the first significant increase in the amount of PCR product.

The time of the increase correlates inversely to the initial amount of DNA template

SYBR green Emits a strong fluorescent signal upon binding to double-

stranded DNA Nonspecific binding is a disadvantage Longer amplicons create a stronger signal

Denaturation Annealing End of Elongation

Detection probes Taqman probes Molecular beacons

Denaturation Annealing Elongation

RT- PCR Amplification can be monitored real-

time No post-PCR processing of products High throughput, low contamination

risk Requirement of 1000-fold less RNA

than conventional assays Most specific, sensitive and

reproducible

PCR applications- medical microbiology

1.Classification of organism based on genetic relatedness (genotyping)

- Amplification - Sequencing - Phylogenetic analysis; multiple

sequence alignment & phylogenetic tree construction.

- MEGA6


Phylogenetic analysis of orientia tsustugamuschi subtypes


2. Identification and confirmation of isolate obtained from culture

- Amplification of (eg.16s rDNA) sequence- Sequencing- BLAST HIT to confirm the organism- If not avail, to be registered as a new strain3. Early detection of pathogens in clinical

specimen- Before the production of antibodies- Able to detect Very low quantity


4. Rapid detection of antibiotic resistance - Helps to administer the right antibiotic 5. Detection of mutations - Mutations would result in the respective

alteration of biochemical activities of microbes. - Mutation analysis will help to study the

characteristics of genes.6. Differentiation of toxigenic from non-toxigenic

strains - By using primers to genes that are responsible for

production of a particular toxin.m


The rRNA is the most conserved (least variable) gene in all cells.

Portions of the rDNA sequence from distantly-related organisms are remarkably similiar. This means that sequences from distantly related organisms can be precisely aligned, making the true differences easy to measure.

Thus the comparison of 16s rDNA sequence can show evolutionary relatedness among microorganisms.

The 16s rDNA sequence has hypervariable regions, where sequences have diverged over evolutionary time. These are often flanked by strongly-conserved regions.

Primers are designed to bind to conserved regions and amplify variable regions.


16s rRNA gene

Molecular diagnosis of genetic disorders

Analysis and characterization of genes abnormalities leading to disease.

Understanding genetic diseases pathogenesis Detection of gene mutation (mutational analysis) Study of genetic diseases pattern of inheritance Diagnosis and screening of genetic diseases Prenatal diagnosis Identification of diseases carrier to help in

genetic and pre-marriage counseling

Molecular diagnosis of genetic disorders

Many genetic diseases are caused by subtle changes in individual genes that cannot be detected by karyotyping.

Traditionally the diagnosis of single-gene disorders has depended on the identification of abnormal gene products (e.g., mutant hemoglobin or enzymes) or their clinical effects, such as anemia or mental retardation

Now it is possible to identify mutations at the level of DNA and offer gene diagnosis for several mendelian disorders.

Applications of PCR in pharmacological research

Quantification of gene expression: Discovery of a new target site for therapeutic intervention .Most of the disease pathologies are associated with an altered gene expression leading to an increase or decrease in the activity of cellular proteins. PCR can be employed to detect very small alterations in cellular mRNA encoding for such proteins. RTPCR

PCR in forensic science Identification purposes Identify crime suspects Exonerate persons wrongly accused of

crime Identify crime and catastrophe victims Establish paternity and other family

relationships

PCR in forensic science RFLP PCR-RFLP HLA-DQ STR analysis Mitochondrial DNA (mtDNA) SNP genotyping VNTRs (variable number of tandem

repeats)

TISSUE TYPING IN TRANSPLANTATIONS

To measure the compatibility of the donor and recipient in transplantation.

The PCR test is a new DNA-based test that can detect the presence or absence of antigens by determining whether cells have the genes for the antigens.

An HLA allele is defined by its entire DNA sequence. There is much sharing of certain polymorphic sequences between different alleles.

Rapid HLA typing is currently best achieved using allele‐specific PCR, whereby DNA primers are used to discriminate between selected sequences in different alleles.

TISSUE TYPING IN TRANSPLANTATIONS

Allele specific PCR in HLA typiny

PCR in medical oncology The gold standard test for cancer diagnosis

of almost all tumors is tissue diagnosis. PCR and/or Southern blot can be used in

diagnosing B and T cell lymphomas. PCR-based detection of T-cell receptor or

immunoglobulin genes rearrangement allow distinction between monoclonal (neoplastic) and polyclonal (reactive) proliferations.

PCR in medical oncology Oncogenes; as proto-oncogenes,

normally promote cell division or cell survival.

Oncogene mutations are usually a gain of function and dominant.

Tumor suppressors: genes normally arrest cell division.

Tumor suppressor gene mutations are usually a loss of function and recessive

Cancer diagnosis…. The EGFR oncogene encodes another of the same

family of epidermal growth factor receptors. This gene is mutated or amplified in several types of

cancer cells. EGFR gene mutations are detected by SSCP, SSP-PCR,

or direct sequencing. The 53-kilodalton tumor suppressor gene encodes a

transcription factor. TP53 is mutated in half of all types of cancer. Loss of TP53 function is an indicator of poor prognosis

in colon, lung, breast, and other cancers. TP53 gene mutations are detected by direct

sequencing.

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Health & Medicine

Polymerase chain reaction