Polymerase chain reactions (PCR) are often performed in searches for the genes of interest, or their messenger RNA transcripts. However, the direct amplification and sequencing of complementary DNA (cDNA) products that encompass 3' and 5' ends have been problematic. The oligo(dT) primer that is traditionally used in the cDNA synthesis step can anneal at any position along the length of a natural (3') or appended (5') poly(A) tail. Subsequently, PCR products of a single, discrete size are not obtained. To enable the acquisition of discrete, first-round PCR products, a “lock-docking” cDNA synthesis primer and the accompanying PCR procedure are described in the chapter. These PCR products can be directly sequenced. This lock-docking primer system may be applied to a novel method for searching previously unidentified gene family members. While PCR targets in the internal, coding regions tend to be of similar lengths, targets in the 3' regions tend to be of varying lengths. Thus, internal PCR products are difficult to separate for further study, while products from 3' regions may be separated by agarose gel electrophoresis.
Bibliographical noteFunding Information:
This work was supported by grants from the National Institutes of Health (NS-27229, LRD) and the American Diabetes Association, Minnesota Affiliate (WLS and LRD).