USE OF INTERSPERSED REPETITIVE SEQUENCES-PCR PRODUCTS FOR CDNA SELECTION

被引：5

作者：

VILLARD, L ^{[1
]}

PASSAGE, E ^{[1
]}

COLLEAUX, L ^{[1
]}

FONTES, M ^{[1
]}

机构：

[1] INSERM,U406,F-13385 MARSEILLE 5,FRANCE

来源：

MAMMALIAN GENOME | 1995年 / 6卷 / 09期

关键词：

D O I：

10.1007/BF00352368

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

In order to increase the efficiency of cDNA selection approaches, we describe the use of interspersed repetitive sequences-PCR (IRS-PCR) products to isolate genes from large-insert genomic clones. IRS-PCR is conducted on total yeast DNA containing a YAC of interest so that there is no need to purify the starting genomic clone. This enables the production of large amounts of genomic substrate for cDNA selection and allows the use of unstable YAC clones. Moreover, the hybridization of the IRS-PCR product to the cDNA clones after selection introduces a positive selection step. We tested these PCR products from YACs for the presence of exons, using cDNAs originating from seven different genes. In each case, at least one exon was present in the IRS-PCR product. We have applied this strategy to four YAC clones originating from the human X Chromosome (Chr). All the selected cDNAs, strongly positive with the LRS-PCR product, did indeed originate from a gene in the region covered by the YAC. In all cases, the previously known genes contained in the genomic clones have been isolated. In addition, we have isolated human genes that have already been described but not assigned to any chromosomal region.

引用

页码：617 / 622

页数：6

共 23 条

[1]

Buckler A.J., Chang D.D., Graw S.L., Brook J.D., Harber D.A., Sharp P.A., Housman D.E., Exon amplification: a strategy to isolate mammalian genes based on RNA splicing, Proc. Natl. Acad. Sci. USA, 88, pp. 4005-4009, (1991)

[2]

Chelly J., Tumer Z., Tonnesen T., Petterson A., Ishikawa-Brush Y., Tommerup N., Horn N., Monaco A.P., Isolation of a candidate gene for Menkes disease that encodes a potential heavy metal binding protein, Nature Genet., 3, pp. 14-19, (1993)

[3]

Cheng S., Fockler C., Barnes W.M., Higuchi R., Effective amplification of long targets from cloned inserts and human genomic DNA, Proc. Natl. Acad. Sci. USA, 91, pp. 5695-5699, (1994)

[4]

Dong B., Horowitz D.S., Kobayashi R., Krainer A.R., Purification and cDNA cloning of HeLa cell p54<sup>nrb</sup>, a nuclear protein with two RNA recognition motifs and extensive homology to human splicing factor PSF and Drosophila NONA/BJ6, Nucleic Acids Res., 21, pp. 4085-4092, (1993)

[5]

Feinberg A.P., Vogelstein B., A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity, Anal. Biochem., 132, pp. 6-13, (1983)

[6]

Gecz J., Villard L., Lossi A.M., Millasseau P., Djabali M., Fontes M., Physical and transcriptional mapping of DXS56-PGK1 1Mb region: identification of three new transcripts, Hum. Mol. Genet., 2, pp. 1389-1396, (1993)

[7]

Gecz J., Polard H., Consalez G., Villard L., Stayton C., Millasseau P., Khrestchatisky M., Fontes M., Cloning and expression of a murine homologue of a putative human X-linked nuclear protein gene closely linked to PGK1 (Xq13.3), Human Molecular Genetics, 3, pp. 39-44, (1994)

[8]

Gibbons R.J., Picketts D.J., Villard L., Higgs D.R., X-linked mental retardation associated with α-thalassaemia (ATR-X syndrome) results from mutations in a putative global transcriptional regulator, Cell, 80, pp. 837-845, (1995)

[9]

Graeber M.B., Kupke K.G., Muller U., Delineation of the dystoniaparkinsonism syndrome locus in Xq13, Proc. Natl. Acad. Sci. USA, 89, pp. 8245-8248, (1992)

[10]

Lengauer C., Green E.D., Cremer T., Fluorescence in situ hybridization of YAC clones after Alu-PCR amplification, Genomics, 13, pp. 826-828, (1992)

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