A reliable PCR amplification method for microdissected tumor cells obtained from paraffin-embedded tissue.

Author(s): Akalu A, Reichardt JK

Publication: Genet Anal, 1999, Vol. 15, Page 229-33

PubMed ID: 10609759 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   Amplification of 959 bp fragments was possible using DNA extracted using QIAquick, but the maximum amplicon size was 309 using DNA that was phenol-chloroform extracted or that did not undergo further extraction after digestion. Further, the PCR productivity was estimated to be 3 times greater using DNA extracted using QIAquick than when DNA extracted with phenol-chloroform was used. Using Platinum Taq led to 3-4 times greater yields of amplified DNA than when AmpliTaq gold was used and 10-20 times more than when Taq polymerase was used.

   Summary of Findings:

   Amplification of 959 bp fragments was possible using DNA extracted using QIAquick, but the maximum amplicon size was 309 bp using DNA that was phenol-chloroform extracted or that did not undergo further extraction after digestion. Further, the PCR productivity was estimated to be 3 times greater using DNA extracted using QIAquick than when DNA extracted with phenol-chloroform was used. Using Platinum Taq for PCR led to 3-4 times greater yields of amplified DNA than when AmpliTaq gold was used and 10-20 times more than when Taq polymerase was used.

   Biospecimens

   • Tissue - Prostate

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Proteinase K digestion and boiling only Phenol chloroform extraction after proteinase K digestion and boiling Modified QIAquick gel extraction kit
   PCR Specific	Targeted nucleic acid	SRD5A2/exon 1 (843-1113) SRD5A2/exon 1 (704-904) SRD5A2/exon 1 (714-1023) HSD3B2/exon 1-2 (1221-1742) HSD3B2/exon 4 (7865¿8824)
   PCR Specific	Length of gene fragment	309 bp 521 bp 959 bp
   PCR Specific	Reaction solution	AmpliTaq gold Platinum Taq Taq polymerase

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