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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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