Ray P F, Handyside A H
Human Embryology Laboratory, Institute of Obstetrics and Gynecology, Royal Postgraduate Medical School Hammersmith Hospital, London, UK.
Methods Mol Med. 1996;5:245-58. doi: 10.1385/0-89603-346-5:245.
The detection of genetic defects in human embryos following in vitro fertilization (IVF) or preimplantation genetic diagnosis (PGD) allows the selection and transfer of unaffected embryos in couples known to be at risk of transmitting an inherited disorder. This avoids the need to termiate an affected pregnancy, following prenatal diagnosis at later stages (1). Diagnosis of a single gene defect is usually performed on one or two single cells (blastomeres) biopsied from 8- to 10-cell embryos on the 3rd d postinsemination using nested polymerase chain reaction (PCR) to amplify informative fragments. Nested PCR allows amplification from a limited number of target sequences (2), and under carefully optimized conditions, amplification of as few as one or two target copies present in a single haploid or diploid cell is possible (3-5). PGD was first achieved for X-linked diseases by determining the sex of the embryos using a Y chromosome-specific repetitive sequence and selective transfer of only female embryos (6). More recently, specific diagnosis has been achieved for cystic fibrosis (CF), by amplifying across the cystic fibrosis transmembrane regulator (CFTR) gene †F508 locus (7) and for Lesch-Nyhan syndrome by amplifying across a familial base substitution nullifying a natural XhoI restriction site in the hypoxanthine phophoribosyl transferase (HPRT) gene (8). In both instances, nested PCR strategies were chosen to amplify the mutated sequence allowing sufficient amplification for detection on ethidium bromide-stained gels. The limited cycling with the outer primers (20 cycles) reduces nonspecific amplification, and only specific fragments that contain the complementary sequence to the internal primers are amplified to a detectable level in the second round of PCR. Although extra handling is involved, any genomic contaminant introduced after the first round of amplification would not be amplified to a detectable level by the inner primers alone. The efficiency of the second amplification is improved because the denaturation of the first amplification product (amplicon) is easier. Also, the great excess of these amplicons compared with nonspecific sequences eliminates competition, thereby enhancing specificity and yield.
在体外受精（IVF）或植入前基因诊断（PGD）后对人类胚胎进行基因缺陷检测，能够在已知有遗传疾病传递风险的夫妇中选择并移植未受影响的胚胎。这样就避免了在孕后期进行产前诊断后终止受影响妊娠的需要（1）。单基因缺陷的诊断通常在授精后第3天从8至10细胞胚胎中活检的一两个单细胞（卵裂球）上进行，使用巢式聚合酶链反应（PCR）扩增有信息价值的片段。巢式PCR能够从有限数量的靶序列进行扩增（2），并且在经过精心优化的条件下，单个单倍体或二倍体细胞中仅有的一两个靶拷贝也有可能被扩增（3 - 5）。PGD首次用于X连锁疾病的诊断，是通过使用Y染色体特异性重复序列确定胚胎性别，并仅选择性移植雌性胚胎（6）。最近，通过扩增囊性纤维化跨膜调节因子（CFTR）基因†F508位点（7）实现了对囊性纤维化（CF）的特异性诊断，以及通过扩增次黄嘌呤磷酸核糖转移酶（HPRT）基因中一个家族性碱基替代使天然XhoI限制性位点无效的序列实现了对莱施 - 奈恩综合征的诊断（8）。在这两种情况下，都选择了巢式PCR策略来扩增突变序列，以便在溴化乙锭染色的凝胶上进行足够的扩增以进行检测。外引物的有限循环次数（20个循环）减少了非特异性扩增，并且只有包含与内引物互补序列的特异性片段在第二轮PCR中被扩增到可检测水平。尽管涉及额外的操作，但第一轮扩增后引入的任何基因组污染物仅靠内引物是不会被扩增到可检测水平的。因为第一轮扩增产物（扩增子）的变性更容易，所以第二轮扩增的效率得到了提高。此外，与非特异性序列相比，这些扩增子的大量过剩消除了竞争，从而提高了特异性和产量。
