Milan D, Riquet J, Yerle M, Goureau A, Schmitz A, Cribiu E P, Frelat G, Gellin J
Laboratoire de Genetique Cellulaire, INRA, Castanet-Tolosan, France.
Mamm Genome. 1996 Mar;7(3):194-9. doi: 10.1007/s003359900053.
Numerous loci can be amplified by PARM-PCR on 300 sorted chromosomes in low-stringency conditions (annealing at 30 degrees C during the two first cycles) to produce a probe that can be used in FISH painting experiments. We demonstrate that, depending on the primer chosen for the amplification, patterns of different quality can be obtained. In order to design a primer that allows amplification of coding sequences, we have shown that motifs of at least seven glutamic acid repeats (GAG or GAA codons) are present in human proteins more frequently than expected. Moreover, these repeats do not correspond to triplet expansion and can be conserved between species. Using probes prepared from sorted chromosomes with (GAG)7 primer, we were able to achieve homologous FISH painting on human, porcine, ovine, and bovine species, and bidirectional heterologous FISH painting between human and porcine species. As an example, using probes for human Chromosome (Chr) 19 and porcine Chrs 1 and 6, we clearly defined the regional homologies existing between those chromosomes.
在低严格条件下（前两个循环在30℃退火），通过PARM-PCR可在300条分选的染色体上扩增出许多基因座，以产生可用于荧光原位杂交（FISH）涂染实验的探针。我们证明，根据用于扩增的引物不同，可以获得不同质量的图谱。为了设计一种能够扩增编码序列的引物，我们发现人类蛋白质中至少含有七个谷氨酸重复序列（GAG或GAA密码子）的基序比预期更为常见。此外，这些重复序列并不对应于三联体扩增，并且在物种间具有保守性。使用由分选染色体与（GAG）7引物制备的探针，我们能够在人类、猪、绵羊和牛物种上实现同源FISH涂染，并在人类和猪物种之间实现双向异源FISH涂染。例如，使用针对人类19号染色体（Chr）以及猪1号和6号染色体的探针，我们清晰地界定了这些染色体之间存在的区域同源性。
