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4 views

PCR

Uploaded by

qadfafasd
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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PCR

PCR or polymerase chain reaction is a diagnostic technique


used to amplify a specific segment of the DNA. A PCR uses DNA
primers to select a segment of the genome and then
nucleotides are used to amplify the DNA count to several
millions or billions via several rounds of DNA synthesis.(Kubista
et al., 2006)

PCR amplifies the DNA count using DNA synthesis that goes
through the same three steps every cycle-

1.Denaturation- This is the first step of PCR, where under


higher temperatures of 94-98 C for 20-30 seconds, double
stranded DNA gets separated into single stranded DNA,
due to the breakage of hydrogen bonds present between
the base pairs.

2.Annealing – The second step of PCR is annealing, where


complementary parts of the primer and the single
stranded DNA i.e. template, is allowed to pair. This process
is temperature sensitive (45-60 C), and the temperature
needs to be maintained as that allows optimal primer
hybridization. Once the primer binds to the template, DNA
polymerase binds to the template-primer hybrid and starts
DNA-synthesis.

3.Extension- The last step of PCR uses a thermostable DNA


polymerase like Taq polymerase, where under 75-80 C, the
polymerase adds nucleotides in the 5’-3’ direction and
synthesizes the complementary strand to the template.

Typically, a PCR cycle is repeated around 20-30 times to acquire


the adequate amount of DNA count.(Kubista et al., 2006)

Quantitative PCR (qPCR)

Quantitative PCR or qPCR or Real-time PCR is a molecular


biology diagnostic technique that is used to quantify the
nucleotide content/ DNA species present in a target sample in
real-time after the amplification of a target DNA sequence. It
uses dyes and probes that bind to the DNA sample and as the
amplification of the DNA sample occurs, the probe / dye sends
signals that are captured in real time, hence the name real-time
PCR.(Ferre, 1992)

qPCR typically uses two types of methods for detection of the


PCR products-

1.Non-Specific Fluorescent dyes that bind with any double


stranded DNA.
2.Sequence specific DNA probes, that contain
oligonucleotides that are labelled with a fluorescent
reporter that allows for the detection, after the binding of
the probe and complementary sequence.
RT-PCR

RT-PCR or Reverse Transcription polymerase chain reaction is a


molecular biology technique used to detect and quantify RNA. It
incorporates reverse transcription of RNA into its
complementary DNA and amplification of specific DNA
segments using PCR.(Jansson & Stone, 2019)

Steps involved in RT-PCR include-

1.Primer design- Primers are designed and synthesized with


respect to the target gene. These primers are
approximately 20-25 bp in length.
2.RNA Extraction- The RNA is extracted from the cells or
tissues.
3.Reverse Transcription- RNA is used to create its
corresponding complementary DNA.
4.Real-time quantification- In Real time, the PCR quantifies
the gene expression by the cDNA by using fluorescent
DNA probes, that enable real time PCR product detection.
5.Result analysis- The fluorescent signal provides a base line
during the first few cycles and as the number of cycles
increase the change in the fluorescent signal is compared
against this baseline for the result analysis.

qRT-PCR

Quantitative Reverse transcriptase polymerase chain


reaction or qRT-PCR is used to amplify the RNA count after
performing reverse transcription to acquire cDNA and then
quantifies the count using dyes and probes, essentially
combining qPCR and RT-PCR procedures into a single process.
(Bustin & Nolan, 2004)

qRT-PCR works on the following principles

1.Reverse Transcription- qRT-PCR begins with the


conversion of RNA to its cDNA.

2.Amplification- The cDNA is then amplified in count using


specifically designed and synthesized primers and DNA
polymerase enzymes.

3.Detection/Quantification- Fluorescent dyes or sequence


specific probes are used to detect the PCR products, by
measuring the emitted fluorescence after each cycle.

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