Types of PCR - WordPress.com · Types of PCR Mitesh Shrestha . PCR - Polymerase Chain Reaction •...

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Types of PCR Mitesh Shrestha

Transcript of Types of PCR - WordPress.com · Types of PCR Mitesh Shrestha . PCR - Polymerase Chain Reaction •...

Types of PCR

Mitesh Shrestha

PCR - Polymerase Chain Reaction

• PCR is an in vitro technique for the amplification of a region of DNA which lies between two regions of known sequence.

• PCR amplification is achieved by using oligonucleotide primers.

– These are typically short, single stranded oligonucleotides which are complementary to the outer regions of known sequence.

• The oligonucleotides serve as primers for DNA polymerase and the denatured strands of the large DNA fragment serves as the template.

– This results in the synthesis of new DNA strands which are complementary to the parent template strands.

Polymerase Chain Reaction

• The basic steps in conducting a conventional PCR involves;

– Denaturation achieved by heating the reaction mixture to a temperature between 90-98° C such that the dsDNA is denatured into single strands by disrupting the hydrogen bonds between complementary bases

– Annealing achieved by cooling the reaction mixture to a temperature of 45-60° C such that the primers base pair with the complementary sequence in the DNA and the hydrogen bonds reform.

– Elongation/ Extension achieved by adjusting the temperature to 72° C which is ideal for polymerase allowing primers extension by joining the bases complementary to DNA strands, the polymerase continually adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template.

• This completes a first cycle another cycle is continued. As PCR machine is automated thermocycler the same cycle is repeated upto 30-40 times

Types of PCR

• INVERSE PCR

• Multiplex PCR

• NESTED PCR

• METHYLATION-SPECIFIC PCR (MSP)

• HOT-START PCR

• ALLELE-SPECIFIC PCR

• HELICASE-DEPENDENT AMPLIFICATION

• REVERSE TRANSCRIPTION PCR (RT-PCR)

• QUANTITATIVE PCR (Q-PCR)

• COLONY PCR

INVERSE PCR

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

• Self-ligation is induced under low concentrations causing the phosphate backbone to reform. This gives a circular DNA ligation product.

• Target DNA is then restriction digested with a known endonuclease. This generates a cut within the known internal sequence generating a linear product with known terminal sequences. This can now be used for PCR (polymerase chain reaction).

• Standard PCR is conducted with primers complementary to the now known internal sequences

Multiplex PCR

• Multiplex PCR is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment.

• In a multiplexing assay, more than one target sequence can be amplified by using multiple primer pairs in a reaction mixture. As an extension to the practical use of PCR, this technique has the potential to produce considerable savings in time and effort within the laboratory without compromising on the utility of the experiment.

• Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis.

NESTED PCR

• Increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA.

• Two sets (instead of one pair) of primers are used in two successive PCRs.

• In the first reaction, one pair of primers “outer pair” 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 after the reaction is diluted with a set of second set “nested or internal” primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction.

• The specificity of PCR is determined by the specificity of the PCR primers.

• For example, if your primers bind to more than one locus (e.g. paralog or common domain), then more than one segment of DNA will be amplified. To control for these possibilities, investigators often employ nested primers to ensure specificity

METHYLATION-SPECIFIC PCR (MSP)

• Methylation-specific PCR (MSP) is used to identify patterns of DNA methylation at cytosine-guanine (CpG) islands in genomic DNA.

• Target DNA is first treated with sodium bisulphite, which converts unmethylated cytosine bases to uracil, which is complementary to adenosine in PCR primers.

• Two amplifications are then carried out on the bisulphite-treated DNA: One primer set anneals to DNA with cytosines (corresponding to methylated cytosine), and the other set anneals to DNA with uracil (corresponding to unmethylated cytosine). MSP used in Q-PCR provides quantitative information about the methylation state of a given CpG island

HOT-START PCR

• Modified form of Polymerase chain reaction (PCR) which avoids a non-specific amplification of DNA by inactivating the taq polymerase at lower temperatures.

• In Hot start PCR specific antibodies are used to block the Taq-polymerase at lower temperature.

• An initial step at 95℃ is required for denaturing the antibodies linked to the active center of the enzyme. The anti-Taq antibodies reduce the Taq polymerase activity below 72℃, the optimal temperature at which the enzyme extends the primers. When the specific antibodies detach from Taq-polymerase, the amplification proceeds with greater specificity.

ALLELE-SPECIFIC PCR

• A diagnostic or cloning technique which is based on single-nucleotide polymorphisms (SNPs) (single-base differences in DNA).

• It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the SNP.

• PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence.

HELICASE-DEPENDENT AMPLIFICATION

• Similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles.

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

REVERSE TRANSCRIPTION PCR (RT-PCR)

• A PCR designed 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. The 5' end of a gene (corresponding to the transcription start site) is typically identified by RACE-PCR ( Rapid Amplification of cDNA Ends).

QUANTITATIVE PCR (Q-PCR)

• Used to measure the quantity of a PCR product (commonly in real-time).

• It quantitatively measures starting amounts of DNA, cDNA or RNA.

• Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample.

• Quantitative real-time PCR has a very high degree of precision.

• QRT-PCR methods use fluorescent dyes, such as Sybr Green, EvaGreen or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time.

• It is also sometimes abbreviated to RT-PCR (Real Time PCR) or RQ-PCR.

COLONY PCR

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

• Selected colonies of bacteria or yeast are picked with a sterile toothpick or pipette tip from a growth (agarose) plate. This is then inserted into the PCR master mix or pre-inserted into autoclaved water. PCR is then conducted to determine if the colony contains the DNA fragment or plasmid of interest

TOUCHDOWN PCR (STEP-DOWN PCR)

• A variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses.

• The annealing temperature at the initial cycles is usually a few degrees (3-5°C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3-5°C) below the primer Tm.

• The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles.

Assignment

• Write short notes on different types of PCR. [7.5]

• Differentiate between Reverse Transcriptase PCR and Real Time PCR. [2.5]