Gabriele A. Zangenberg, Ph.D. and Rebecca Reynolds, Ph.D.
Roche Molecular Systems, Inc., Alameda, CA

× Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø

Generally, the PCR process is designed to amplify a single specific target sequence in one reaction tube. However, the need for simultaneous amplification of multiple targets in a single PCR is increasing (e.g., the identification of several pathogens in a biological sample, mutational analysis of multiple gene exons, and simultaneous interrogation of multiple loci for human genetic linkage or oncogenic studies). We are particularly interested in the co-amplification of multiple polymorphic markers for forensic casework, which simplifies the experimental setup, saves considerable amounts of sample DNA and time, and also significantly reduces cost. The first multiplex PCR system used for forensic DNA typing was the AmpliType PM^tm kit. Using this kit, six genetic markers (HLA DQA1, LDLR, GYPA, HBGG, D7S8, GC) are amplified in a single reaction, from as little as 1 ng extracted DNA. Five of the six markers (excluding HLA DQA1) are typed simultaneously, using a single immobilized sequence-specific oligonucleotide (SSO) probe strip. The combined probability of inclusion for these five markers is ~ 1 in 5000. Combining 18 genetic markers, including ABO and gender, decreases the probability that two unrelated individuals will have the same types to only 1 in 1 billion.

Optimization studies of the PM multiplex amplification reaction were carried out for nearly three years before acceptable PCR product specificity and yield were achieved. Some of the problems encountered when developing a multiplex PCR system are excessive primer-dimer, loss of specificity, and decreased yield of particular PCR products. Fortunately, numerous improvements to multiplex PCR have been made, including improved primer design, new thermostable DNA polymerases, and modified reaction conditions (e.g., "hot start" PCR). AmpliTaq Gold^tm provides "hot start" performance, which is essential when co-amplifying multiple gene sequences, and significantly reduces the time required to optimize the PCR conditions. We have optimized a PCR system which allows the co-amplification of up to 34 different gene sequences. Studies with our new formulation have shown that low copy number targets as well as difficult targets can be amplified very specifically with negligible amounts of primer-dimer.