Polymerase chain reaction uses

PCR Uses

Principle of the test

·    Molecular method used to amplify DNA sequence of interest (creating a maximum of 2n double-stranded DNA molecules per cycle of replication) that can be used to diagnose conditions, identify mutations, identify individuals

·    DNA sample is placed in a PCR reaction containing Taq polymerase, appropriate buffers and cations (eg. magnesium) required for the function of the Taq enzyme, dNTPs, and primers complementary to 5’ and 3’ ends of the sequence of interest

·    Reaction is put through several cycles of denaturation, annealing and elongation (at various temperatures) to exponentially amplify the sequence of interest

·    The PCR product is then run on an agarose/polyacrylamide gel against known controls to determine whether or not the DNA contains the sequence of interest

Polymerase chain reaction PCR applications as a diagnostic tool in the laboratory

·    Amplification of TCR, IgH, Jκ chains to determine T-cell or B-cell clonality in lymph nodes to diagnose lymphomas

·    Determination of presence or absence of TB in tissues of interest

·    Identification of individuals in forensics cases

·    Identification of translocations (eg. EWS, CML, etc.)

·    Identification of single gene disorders

·    Determination of MSI

Polymerase chain reaction (PCR)

Polymerase chain reaction technique

Polymerase chain reaction is a molecular biology technique and method used to amplify a DNA sequence of interest (creating a maximum of 2n double-stranded DNA molecules per cycle of replication) that can be used to diagnose conditions, identify mutations, identify individuals

The DNA sample is placed in a PCR reaction containing Taq polymerase, appropriate buffers and cations (eg. magnesium) required for the function of the Taq enzyme, dNTPs, and primers complementary to 5’ and 3’ ends of the sequence of interest

The reaction is put through several cycles of denaturation, annealing and elongation (at various temperatures) to exponentially amplify the sequence of interest

The PCR product is then run on an agarose/polyacrylamide gel against known controls to determine whether or not the DNA contains the sequence of interest

Polymerase chain reaction applications as a diagnostic tool

•    Amplification of T-cell receptor gene (TCR), IgH, Jκ chains to determine T-cell or B-cell clonality in lymph nodes to diagnose lymphomas

•    Determination of presence or absence of TB in tissues of interest

•    Identification of individuals in forensics cases

•    Identification of translocations (eg. EWS, CML, etc.)

•    Identification of single gene disorders

•    Determination of microsatellite instability (MSI)

Polymerase chain reaction problems and troubleshooting

•    Contamination of sample can create false positives

•    Degenerate (non-specific) primers can create false positives

•    Lack of stringency with regard to annealing temperatures (false positives)

•    Suboptimal temperatures for Taq polymerase function (false negatives)

•    Omission of components of reaction (false negatives)