Mol Bio Pcr Variants

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process that involves a reverse transcriptase (RTase), an enzyme that uses RNA as the template to make complementary DNA (cDNA)

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Reverse Transcription PCR

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Enzyme in RT PCR

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Reverse Transcriptase

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the exact opposite of the naturally occurring DNA transcription in which RNA is synthesized using DNA as the template

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Reverse Transcription

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two-step procedure of RT PCR

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-Reverse Transcription

-Amplification

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Application of RT PCR

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-Identify RNA Genomes (Viruses)

-Gene Expression

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2 activities of RT PCR

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-Polymerase Activity

-Nuclease Activity

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Nuclease Activity

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RNAse H

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(RNA Dependent DNA Polymerase

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Polymerase Activity

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used to amplify multiple target sequences in a single PCR reaction using multiple primer sets

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Multiplex PCR

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Multiplex PCR is used to detect

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-Mutations

-Deletions

-Polymorphism

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Multiplex PCR used to detect different viral, bacterial and other pathogens in a

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Single Tube

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consumes less time and effort in obtaining the results

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Multiplex PCR

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different bands of DNA products

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Multiplex PCR

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Multiplex was used to detect different----- in a single tube

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-Bacterial

-Viral

-Some Pathogens

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a modification of PCR that was designed to improve sensitivity and specificity

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Nested PCR

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For RNA Genomes or mRNA gene expression

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Reverse Transcription PCR

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Multiple sets of primers and target strands while having a singe PCR reaction

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Multiplex PCR

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PCR was done 2 times

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Nested PCR

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involves the use of two primer sets and two successive PCR reactions

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Nested PCR

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Nested PCR involves the use of

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-Two Primer Sets

-Two Successive PCR reactions

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There are how many cycles in nested pcr

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2

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Primers in nested PCR

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-External Primers

-Nested Primers

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Primers in the 1st cycle

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External Primers

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Primers in the 2nd Cycle

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Nested Primers

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anneal to sequences upstream from the second set of primers

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External Primers

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Primer that Complementary to the target sequence of Nested PCR

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Nested primer

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Anneals within the target sequence

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Nester Primer

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qPCR is also called as

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Real-time PCR

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Meaning of q in qPCR

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Quantitative

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monitors the amplification of a targeted DNA molecule during the PCR

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qPCR

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Do qPCR needs electrophoresis

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No

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In qPCR what is the relationship between the Fluorescence and Concentration of PCR products

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Direct Relationship

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Known to quantify and amplify at the same time

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qPCR

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Relative Quantification

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qPCR

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Two common methods for the detection of PCR products in real-time PCR are:

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-Non-specific Fluorescent Dyes

-Sequence-Specific DNA probe

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intercalate with any double-stranded DNA

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Non-specific Fluorescent Dye

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consisting of oligonucleotides that are labelled with a fluorescent reporter, which permits detection only after hybridization of the probe with its complementary sequence

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Sequence-Specific DNA Probe

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Example of Non-specific Fluorescent Dye

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Sybr Green

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short segment of nucleic acid

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DNA probe

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Dyes of DNA probe

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-Reporter Dye

-Quencher Dye

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Most common brand of probe

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Taqman Probe

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Fluorescent dye of the DNA Probe

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Reporter Dye

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Non-fluorescent dye of the DNA Probe

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Quencher Dye

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In qPCR the DNA polymerase possess

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Exonuclease Activity

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✓Separates reporter and quencher dyes Amount of fluorescence captured by the machin

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Exonuclease Activity

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What variant of PCR does stops in every 10 cycles

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qPCR

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When do qPCR stops

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Every 10 cycle

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enables the absolute and reproducible quantification of target nucleic acids

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Digital PCR

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What is more superior dPCR or qPCR

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dPCR

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the initial sample mix is partitioned into a large number of individual wells prior to the amplification step, resulting in either 1 or 0 targets being present in each well

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Digital PCR

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How many targets are present in the wells of dPCR

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1-0

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measures the amount of DNA after amplification is complete and then determines the fraction of replicates.

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Digital PCR

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Absolute Quantification

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Digital PCR

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in nanovolume - there are 1 to 2 templates of DNA

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Wells

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Wells are found in

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Digital PCR

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wells with no products

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Negative Control

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the target sequence is amplified at a constant temperature of 60–65 °C

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LAMP PCR

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Temperature of LAMP PCR

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60-65 C

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Variant of PCR that does not need denaturation

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LAMP PCR

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Cheapest PCR

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LAMP PCR

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There are how many primers in LAMP PCR

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4

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Isothermal

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LAMP PCR

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DNA Polymerase of LAMP PCR

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BST DNA Polymerase

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Meaning of BST in BST DNA polymerase

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Bacillus Stearothermophillus

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BST DNA Polymerase Activity

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-Polymerase Activity

-DNA Displacement Activity

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4 primers of LAMP PCR

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-F3

-FIP

-B3

-BIP

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✓unzips DNA duplex before synthesis

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DNA Displacement Activity

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Disadvantage of LAMP PCR

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Primer Design (Many Primer)