Detection of mutations in human type I collagen mRNA in osteogenesis imperfecta by indirect RNAase protection

C. Genovese, A. Brufsky, Jay Shapiro, D. Rowe

Research output: Contribution to journalArticle

Abstract

A method for detecting a wide variety of mutations within type I collagen has been developed and evaluated on a series of patients with osteogenesis imperfecta. RNA, extracted from the nuclear and cytoplasmic compartment of cultured fibroblasts from affected individuals, is hybridized with antisense single-stranded cDNA to the α1(I) mRNA. The hybrid is digested with RNAase A and T1 under varying degrees of stringency. The resistant RNA bands are separated by electrophoresis in agarose, transferred to nitrocellulose, and hybridized with antisense cRNA colinear with the protecting probe. This approach is capable of detecting previously defined mutations such as 252-base pair deletion and a 1-base pair mismatch within the α1(I) mRNA. The method appears to be particularly useful in detecting abnormalities of RNA processing that behave as an insert or deletion within the mature mRNA. The procedure should be generally applicable for the identification and localization of any mutation within an entire gene if the gene is expressed as an RNA and a complete cDNA for the mRNA is available.

Original languageEnglish (US)
Pages (from-to)9632-9637
Number of pages6
JournalJournal of Biological Chemistry
Volume264
Issue number16
StatePublished - 1989
Externally publishedYes

Fingerprint

Osteogenesis Imperfecta
Collagen Type I
RNA
Messenger RNA
Mutation
Base Pair Mismatch
Complementary DNA
Genes
Nuclear RNA
Complementary RNA
Collodion
Fibroblasts
Electrophoresis
Base Pairing
Sepharose
Processing

ASJC Scopus subject areas

  • Biochemistry

Cite this

Detection of mutations in human type I collagen mRNA in osteogenesis imperfecta by indirect RNAase protection. / Genovese, C.; Brufsky, A.; Shapiro, Jay; Rowe, D.

In: Journal of Biological Chemistry, Vol. 264, No. 16, 1989, p. 9632-9637.

Research output: Contribution to journalArticle

@article{f635accef8fb4e40b215ceb79d31d401,
title = "Detection of mutations in human type I collagen mRNA in osteogenesis imperfecta by indirect RNAase protection",
abstract = "A method for detecting a wide variety of mutations within type I collagen has been developed and evaluated on a series of patients with osteogenesis imperfecta. RNA, extracted from the nuclear and cytoplasmic compartment of cultured fibroblasts from affected individuals, is hybridized with antisense single-stranded cDNA to the α1(I) mRNA. The hybrid is digested with RNAase A and T1 under varying degrees of stringency. The resistant RNA bands are separated by electrophoresis in agarose, transferred to nitrocellulose, and hybridized with antisense cRNA colinear with the protecting probe. This approach is capable of detecting previously defined mutations such as 252-base pair deletion and a 1-base pair mismatch within the α1(I) mRNA. The method appears to be particularly useful in detecting abnormalities of RNA processing that behave as an insert or deletion within the mature mRNA. The procedure should be generally applicable for the identification and localization of any mutation within an entire gene if the gene is expressed as an RNA and a complete cDNA for the mRNA is available.",
author = "C. Genovese and A. Brufsky and Jay Shapiro and D. Rowe",
year = "1989",
language = "English (US)",
volume = "264",
pages = "9632--9637",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "16",

}

TY - JOUR

T1 - Detection of mutations in human type I collagen mRNA in osteogenesis imperfecta by indirect RNAase protection

AU - Genovese, C.

AU - Brufsky, A.

AU - Shapiro, Jay

AU - Rowe, D.

PY - 1989

Y1 - 1989

N2 - A method for detecting a wide variety of mutations within type I collagen has been developed and evaluated on a series of patients with osteogenesis imperfecta. RNA, extracted from the nuclear and cytoplasmic compartment of cultured fibroblasts from affected individuals, is hybridized with antisense single-stranded cDNA to the α1(I) mRNA. The hybrid is digested with RNAase A and T1 under varying degrees of stringency. The resistant RNA bands are separated by electrophoresis in agarose, transferred to nitrocellulose, and hybridized with antisense cRNA colinear with the protecting probe. This approach is capable of detecting previously defined mutations such as 252-base pair deletion and a 1-base pair mismatch within the α1(I) mRNA. The method appears to be particularly useful in detecting abnormalities of RNA processing that behave as an insert or deletion within the mature mRNA. The procedure should be generally applicable for the identification and localization of any mutation within an entire gene if the gene is expressed as an RNA and a complete cDNA for the mRNA is available.

AB - A method for detecting a wide variety of mutations within type I collagen has been developed and evaluated on a series of patients with osteogenesis imperfecta. RNA, extracted from the nuclear and cytoplasmic compartment of cultured fibroblasts from affected individuals, is hybridized with antisense single-stranded cDNA to the α1(I) mRNA. The hybrid is digested with RNAase A and T1 under varying degrees of stringency. The resistant RNA bands are separated by electrophoresis in agarose, transferred to nitrocellulose, and hybridized with antisense cRNA colinear with the protecting probe. This approach is capable of detecting previously defined mutations such as 252-base pair deletion and a 1-base pair mismatch within the α1(I) mRNA. The method appears to be particularly useful in detecting abnormalities of RNA processing that behave as an insert or deletion within the mature mRNA. The procedure should be generally applicable for the identification and localization of any mutation within an entire gene if the gene is expressed as an RNA and a complete cDNA for the mRNA is available.

UR - http://www.scopus.com/inward/record.url?scp=0024327710&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024327710&partnerID=8YFLogxK

M3 - Article

C2 - 2542316

AN - SCOPUS:0024327710

VL - 264

SP - 9632

EP - 9637

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 16

ER -