Takahashi N, Ko M S
Furusawa MorphoGene Project, ERATO, Research Development Corporation of Japan (JRDC), Tsukuba.
Genomics. 1993 Apr;16(1):161-8. doi: 10.1006/geno.1993.1153.
Ideal markers for a complementary DNA (cDNA) map of the mouse genome should be amplifiable by the polymerase chain reaction (PCR) and they should be polymorphic for genetic mapping, as well as unique for physical mapping. In our search for such markers, we did comparative sequencing of PCR-amplified genomic DNAs derived from 15 inbred strains and found that about 250 bp of the 3'-end region (3'-ER) of cDNAs, which is the sequence immediately upstream from the poly(A) tail, was polymorphic enough to distinguish the allele of a laboratory strain from that of a wild strain (Mus spretus) in 14 of 22 cDNAs tested. Specifically, in 9 of these 14 cDNAs, each allele was identified by the restriction fragment length polymorphism. This data indicates that about 65% of the 3'-ERs of cDNAs can be automatically transformed into PCR-based genetic markers named "biallelic polymorphic expressed sequence tags (bESTs)." These markers can be easily and precisely mapped by typing of the panels of interspecific backcrosses. Because a large number of bEST markers can be efficiently obtained by a single-run automated sequencing of randomly selected cDNAs, these markers will greatly facilitate the construction of high-resolution genetic and physical maps of expressed sequences of the mouse genome.
小鼠基因组互补DNA（cDNA）图谱的理想标记应能通过聚合酶链反应（PCR）扩增，且应具有多态性以便进行遗传图谱绘制，同时在物理图谱绘制中具有唯一性。在寻找此类标记的过程中，我们对来自15个近交系的PCR扩增基因组DNA进行了比较测序，发现cDNA 3'端区域（3'-ER）约250 bp的序列，即紧接在poly(A)尾上游的序列，其多态性足以在22个测试的cDNA中的14个中区分实验室品系和野生品系（小家鼠）的等位基因。具体而言，在这14个cDNA中的9个中，每个等位基因可通过限制性片段长度多态性来鉴定。这些数据表明，约65%的cDNA的3'-ER可自动转化为基于PCR的遗传标记，即“双等位基因多态性表达序列标签（bESTs）”。通过对种间回交群体进行分型，这些标记能够轻松且精确地进行图谱绘制。由于通过对随机选择的cDNA进行单次自动测序就能高效获得大量的bEST标记，这些标记将极大地促进小鼠基因组表达序列的高分辨率遗传图谱和物理图谱的构建。
