Method to Identify Biases in PCR Amplification of T-Cell Receptor Variable Region Genes

摘要

Quantitative polymerase chain reaction (PCR) methods have been used to establish the relative frequencies of expression of T-cell receptor (TCR) beta (B) and alpha (A) variable (V) gene families in vitro and in various tissues in vivo. This quantitative approach is difficult as it is influenced by factors inherent in the PCR, and quantitation must be performed during the exponential phase of DNA amplification (8). An additional potential problem is that variation in the amplification efficiencies ofdifferent TCR segments is possible with several of the commonly used PCR methods (8). If these amplification biases are not corrected for, they could lead to spurious conclusions as to relative TCR segment expression levels in blood or in another tissuefrom an individual. Some investigators (1,5) have assumed that amplification biases do not occur and that different primer pairs amplify different TCRV gene segments with equal efficiencies. Others, aware of the potential problem, have compared the relative levels of expression of particular TCR segments between individuals or between tissues in an individual (3). In a variation of this latter approach, Lovebridge et al. (6) controlled for the amounts of starting TCRB constant (C) region templates and then compared TCRBV gene expression between different individuals. No quantitative PCR method reliably establishes the relative expression levels of different TCR segments in a tissue from a particular individual. We report a simple method to do this. Themethod assesses the relative efficiencies of priming of TCRBV (BV) segments when using a set of sequence-specific oligonucleotide (SSO) BV primers, and we describe a method to correct for amplification biases.