瓊脂糖凝膠與聚丙烯酰胺凝膠
瓊脂糖 . 長度彼此相差 30–40bp 的多重 PCR 產物在通常使用的 SeaKem 或 NuSieve(FMC BioProducts) 3% 的瓊脂糖凝膠可以很好的區分開。在低電場強度下過夜電泳可以優化每個 PCR 產物條帶的電泳效果，特別是當 PCR 產物小于 400–500 bp 時。
聚丙烯酰胺凝膠 . 為了分離長度僅差幾個 bp 的 PCR 產物（例如微衛星標記）需要使用濃度 6%–10% 的聚丙烯酰胺凝膠 (PAA 膠 ) 。非變性 PAA 膠可以有效的區分非多態性基因，但在這種膠上分離微衛星片斷會出現不正常條帶。例如在分析含有單拷貝人類第 12 號染色體的兩個雜交瘤細胞系的人類第 12 號染色體上某個多態性位點時，對于每一個被檢測的基因位點，在非變性 PAA 膠上都出現了兩個條帶 (e.g., Figure 5d) 。在常規的 6% PAA/7 M urea 測序膠上，每個被檢測的基因位點都僅有一條帶 ( 比較 Figure 5, d 和 e 的產物 ) 。
我們用一系列樣品檢測了多個參數來優化多重 PCR 。對于任何 PCR 反應，為了獲得高特異性的擴增產物，退火溫度和 KCl 濃度（鹽濃度）的最優組合是最重要的。 Mgcl 2 濃度應當與 dNTP 的量成比例，對于任何反應這個比值可以是常數。雖然逐漸提高 MgCL 2 濃度可以增強反應的特異性，但是看起來退火溫度和 KCl 濃度（鹽濃度）對于獲得高產量的特異性 PCR 擴增產物更為重要。在多重 PCR 中調節每一個基因的引物量也很重要。 Figure 1 給出了進行高效的多重 PCR 的有效方法。盡管無法完全列出影響 PCR 反應的所有因素。然而本研究中所提到的參數的優化提供了解決多重 PCR 中常見問題的基本方法。
參考文獻
? Abbs, S. and M. Bobrow. Analysis of quantitative PCR for the diagnosis of deletion and duplication carriers in the dystrophin gene. (1992) J. Med. Genet. 29 , 191-196.
? Anonymous.. Diagnosis of Duchenne and Becker muscular dystrophies by polymerase chain reaction. A multicenter study. (1992) JAMA 267, 2609-2615.
? Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith and K. Struhl. 1994. The polymerase chain reaction, p. 15.1.1. -15.1.4. In K. Janssen (Ed.), Current Protocols in Molecular Biology, Vol.2. Greene Publishing Associates and John Wiley & Sons, New York.
? Beggs, A.H., M. Koenig, F.M. Boyce and L.M. Kunkel. Detection of 98% of DMD/BMD gene deletions by polymerase chain reaction. (1990) Hum. Genet . 86, 45-48.
? Chamberlain, J.S., R.A. Gibbs, J.E. Ranier and C.T. Caskey. 1990. Multiplex PCR for the diagnosis of Duchenne muscular dystrophy,p. 272- 281. In M.A. Innis, D.H. Gelfand, J.J. Sninsky and T.J. White (Eds.), PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego.
? Chamberlain, J.S., R.A. Gibbs, J.E. Ranier, P.N. Nguyen and C.T. Caskey. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. (1988) Nucleic Acids Res. 16 , 11141- 11156.
? Crisan, D. Molecular diagnostic testing for determination of myeloid lineage in acute leukemias. (1994) Ann. Clin. Lab. Sci. 24, 355-363.
? Henegariu, O., P. Hirschmann, K. Kilian, S. Kirsch, C. Lengauer, R. Maiwald, K. Mielke and P. Vogt. Rapid screening of the Y chromosome in idiopathic sterile men, diagnostic for deletions in AZF, a genetic Y factor expressed during spermatogenesis. (1994) Andrologia 26, 97-106.
? Innis, M.A. and D.H. Gelfand. 1990. Optimization of PCRs, p 3-13. In M.A. Innis, D.H. Gelfand, J.J. Sninsky and T.J. White (Eds.), PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego.
? Mansfield, E.S., J.M. Robertson, R.V. Lebo, M.Y. Lucero, P.E. Mayrand, E. Rappaport, T. Parrella, M. Sartore, S. Surrey and P. Fortina. Duchenne/Becker muscular dystrophy carrier detection using quantitative PCR and fluorescence-based strategies. (1993) Am. J. Med. Genet. 48, 200-208.
? Mutirangura, A., F. Greenberg, M.G. Butler, S. Malcolm, R.D. Nicholls, A. Chakravarti and D.H. Ledbetter. Multiplex PCR of three dinucleotide repeats in the Prader-Willi/Angelman critical region (15q11-q13): molecular diagnosis and mechanism of uniparental disomy. (1993) Hum. Mol. Genet. 2 , 143-151.
? Saiki, R.K. 1989. Optimization of the polymerase chain reaction, p. 25-30. In H.A. Erlich, R. Gibbs and H.H. Kazazian Jr. (Eds.), Current Communications in Molecular Biology. CSH Laboratory Press, Cold Spring Harbor, NY.
? Saiki, R.K. 1989. The design and optimization of the PCR, p. 7-16. In H.A. Erlich (Ed.), PCR Technology: Principles and Applications for DNA Amplification. Stockton Press, New York.
? Shuber, A.P., J. Skoletsky, R. Stern and B.L. Handelin. Efficient 12-mutation testing in the CFTR gene: a general model for complex mutation analysis. (1993) Hum. Mol. Genet. 2, 153-158.
? Vandenvelde, C., M. Verstraete and D. Van Beers. Fast multiplex polymerase chain reaction on boiled clinical samples for rapid viral diagnosis. (1990) J. Virol. Methods 30 , 215-227.
Vogt, P.H., A. Edelmann, S. Kirsch, O. Henegariu, P. Hirschmann, P. Kiesewetter, F.M. K?hn, W.B. Schill et al. Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. (1996) Hum. Mol. Genet. 5 , 933-943.