Construction of a full-length murine proα2(I) collagen cDNA by the polymerase chain reaction

Charlotte L. Phillips, Laura W. Lever, Sheldon R. Pinnell, Leigh Quarles, Richard J. Wenstrup

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Construction of large collagen cDNA has been hindered by the relatively large size and high G-C content of processed mRNA. We describe here the development of a rapid and efficient method for obtaining large full-length collagen cDNA. A full-length (4.3 kb) murine proα2(I) collagen cDNA was constructed by synthesis of a first-strand cDNA library with use of poly-A RNA (MC3T3-E1) and the oligo-dT,7-adapter primer described by Frohman et al (Proc Natl Acad Sci USA 85:8998, 1988). Proα2(I) collagen cDNA were specifically amplified by the polymerase chain reaction (PCR) with a proα2(I) specific primer as the 5′ primer (20mer; corresponding to nucleotide positions 42-61 in the first exon of the murine proα2(I) collagen gene, COLLA2), and with the adapter sequence 5′ to the dT17 as the 3′ primer. The PCR conditions were optimized to allow amplification of the expected 4.0-5.0-kb product; a major 4.3-kb product was visualized by ethidium bromide, identified by in situ gel hybridization, and cloned. DNA sequencing determined that it contained the correct 5′ sequence and the 3′ end had a 68 basepair (bp) 3′ untranslated region. The entire sequence that codes the amino-terminal propeptide domain has been determined and compared to the human sequence. The homology between human and mouse is less in the amino terminal propeptide than in the triple helical domain; exon 5 of murine COLLA2 codes for an additional six amino acids not found in human COLLA2.

Original languageEnglish (US)
Pages (from-to)980-984
Number of pages5
JournalJournal of Investigative Dermatology
Volume97
Issue number6
DOIs
StatePublished - Jan 1 1991
Externally publishedYes

Fingerprint

Polymerase chain reaction
Collagen
Complementary DNA
Polymerase Chain Reaction
Exons
Messenger RNA
Ethidium
Base Composition
3' Untranslated Regions
Gene Library
DNA Sequence Analysis
In Situ Hybridization
Amplification
Nucleotides
Genes
Gels
Amino Acids
DNA

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Dermatology
  • Cell Biology

Cite this

Construction of a full-length murine proα2(I) collagen cDNA by the polymerase chain reaction. / Phillips, Charlotte L.; Lever, Laura W.; Pinnell, Sheldon R.; Quarles, Leigh; Wenstrup, Richard J.

In: Journal of Investigative Dermatology, Vol. 97, No. 6, 01.01.1991, p. 980-984.

Research output: Contribution to journalArticle

Phillips, Charlotte L. ; Lever, Laura W. ; Pinnell, Sheldon R. ; Quarles, Leigh ; Wenstrup, Richard J. / Construction of a full-length murine proα2(I) collagen cDNA by the polymerase chain reaction. In: Journal of Investigative Dermatology. 1991 ; Vol. 97, No. 6. pp. 980-984.
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