Lymphoma - B cell neoplasms - Polymerase chain reaction (PCR)

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Lymphoma - B cell neoplasms

Molecular analysis - polymerase chain reaction (PCR)

Reviewer: Nikhil Sangle, M.D., University of Utah and ARUP Laboratories (see Reviewers page)
Revised: 27 February 2011, last major update February 2011
Copyright: (c) 2001-2011, PathologyOutlines.com, Inc.

See also Molecular Pathology chapter

General
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● 10,000 times more sensitive than Southern blot
● Clones appear as 1-2 bands (if 1 or 2 alleles were rearranged); polyclonal cells appear as a smear since no amplification usually occurs due to wide separation of V and J regions
● Faster than Southern blot and minimal tissue required

Uses
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● Helpful for determining clonality of lymphoid aggregates in bone marrow biopsies (Arch Pathol Lab Med 2000;124:511)

Procedure
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● (1) Add sample DNA, 4 nucleotides, buffer with magnesium, primers and Taq DNA polymerase to test tube
● (2) Use PCR cycler with 3 reactions: (a) denature double stranded DNA, (b) allow annealing of primers, (c) allow DNA polymerase activity to extend primer
● (3) Use multiple cycles, to provide exponential expansion of DNA
● (4) Analyze products by gel electrophoresis, then stain gel with ethidium bromide OR transfer gel to nylon membrane by Southern blotting and hybridize membrane with labeled probe
● False negatives are due to imperfect consensus primers, inability to detect partial DJ rearrangements; mutations of Ig genes that prevent annealing of primers; lymphomas that arise from B cell precursors prior to rearrangement

PCR + temperature gradient gel electrophoresis
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● Used to analyze T cell receptor gene rearrangement (Arch Pathol Lab Med 2001;125:202)
● This form of electrophoresis uses temperature gradient across a gel

Reverse transcriptase PCR (RT-PCR)
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● Used to detect chimeric fusion mRNA transcripts translocations
● Highly sensitive technique by which a very low copy number of RNA molecules can be detected

End of Lymphoma - B cell neoplasms > Molecular analysis - Polymerase chain reaction (PCR)

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