Wu G, Su S, Bird R C
Department of Pathobiology, Auburn University, AL 36849-5519.
Genet Anal Tech Appl. 1994;11(2):29-33. doi: 10.1016/1050-3862(94)90057-4.
The efficiency of subtraction, integrity of residual single-stranded cDNA, and efficient recovery of nanogram quantities of double-stranded cDNA are the three most important factors affecting quality of subtractive hybridization reactions prior to subtractive cDNA library construction. Techniques for efficient isolation of single-stranded cDNA, after subtraction, have greatly improved from early protocols based on hydroxylapatite chromatography to phenol-chloroform extraction of biotin-streptavidin-crosslinked polynucleotides or oligo(dA)-cellulose affinity chromatography. Factors affecting mRNA stability at the hybridization step, however, also have consequences that directly affect the complexity of the library and the length of cDNAs recovered. We have optimized the subtractive hybridization step in subtractive cDNA library construction to ensure that single-stranded cDNAs survive hybridization as near to full length as possible. These improvements have enabled successful construction of subtractive cDNA libraries from the nanogram quantities of single-stranded cDNA remaining after extensive liquid hybridization to high calculated C(o)t values.
扣除效率、残留单链cDNA的完整性以及纳克级双链cDNA的高效回收，是影响扣除cDNA文库构建前扣除杂交反应质量的三个最重要因素。扣除后高效分离单链cDNA的技术，已从早期基于羟基磷灰石色谱法的方案，大幅改进为生物素-链霉亲和素交联多核苷酸的酚-氯仿萃取法或寡聚（dA）-纤维素亲和色谱法。然而，杂交步骤中影响mRNA稳定性的因素，也会直接影响文库的复杂性和回收的cDNA长度。我们已优化了扣除cDNA文库构建中的扣除杂交步骤，以确保单链cDNA在杂交后尽可能以接近全长的形式留存。这些改进使得在与高计算C（o）t值进行广泛液相杂交后，从剩余的纳克级单链cDNA成功构建出扣除cDNA文库。
