Properties of targeted preamplification in DNA and cDNA quantification

• Objective: Quantification of small molecule numbers often requires preamplification to generate enough copies for accurate downstream enumerations. Here, we studied experimental parameters in targeted preamplification and their effects on downstream quantitative real-time PCR (qPCR). Methods: To evaluate different strategies, we monitored the preamplification reaction in real-time using SYBR Green detection chemistry followed by melting curve analysis. Furthermore, individual targets were evaluated by qPCR. Result: The preamplification reaction performed best when a large number of primer pairs was included in the primer pool. In addition, preamplification efficiency, reproducibility and specificity were found to depend on the number of template molecules present, primer concentration, annealing time and annealing temperature. The amount of nonspecific PCR products could also be reduced about 1000-fold using bovine serum albumin, glycerol and formamide in the preamplification. Conclusion: On the basis of our findings, we provide recommendations how to perform robust and highly accurate targeted preamplification in combination with qPCR or next-generation sequencing.

Subject headings

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine (hsv//eng)

Keyword

experimental design

multiplex PCR

preamplification

primer-pools

quantitative real-time PCR

REAL-TIME PCR

POLYMERASE-CHAIN-REACTION

BOVINE SERUM-ALBUMIN

EMBRYONIC STEM-CELLS

SINGLE-CELL

GENE-EXPRESSION

QUANTITATIVE PCR

RT-PCR

AMPLIFICATION

BETAINE

Pathology

EVET E

1995

NUCLEIC ACIDS RESEARCH

V23

P3343

ENG S

1994

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF

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