Biomaterial Database

Construction of a full-length murine pro alpha 2(I) collagen cDNA by the polymerase chain reaction. 1991

C L Phillips, and L W Lever, and S R Pinnell, and L D Quarles, and R J Wenstrup

Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.

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 pro alpha 2(I) collagen cDNA was constructed by synthesis of a first-strand cDNA library with use of poly-A RNA (MC3T3-E1) and the oligo-dT17-adapter primer described by Frohman et al (Proc Natl Acad Sci USA 85:8998, 1988). Pro alpha 2(I) collagen cDNA were specifically amplified by the polymerase chain reaction (PCR) with a pro alpha 2(I) specific primer as the 5' primer (20mer; corresponding to nucleotide positions 42-61 in the first exon of the murine pro alpha 2(I) collagen gene, COL1A2), 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 COL1A2 codes for an additional six amino acids not found in human COL1A2.

UI	MeSH Term	Description	Entries
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D011347	Procollagen	A biosynthetic precursor of collagen containing additional amino acid sequences at the amino-terminal and carboxyl-terminal ends of the polypeptide chains.	Protocollagen,Procollagen Type M
D003001	Cloning, Molecular	The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.	Molecular Cloning
D004247	DNA	A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine).	DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
D005784	Gene Amplification	A selective increase in the number of copies of a gene coding for a specific protein without a proportional increase in other genes. It occurs naturally via the excision of a copy of the repeating sequence from the chromosome and its extrachromosomal replication in a plasmid, or via the production of an RNA transcript of the entire repeating sequence of ribosomal RNA followed by the reverse transcription of the molecule to produce an additional copy of the original DNA sequence. Laboratory techniques have been introduced for inducing disproportional replication by unequal crossing over, uptake of DNA from lysed cells, or generation of extrachromosomal sequences from rolling circle replication.	Amplification, Gene
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D001483	Base Sequence	The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.	DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D013698	Templates, Genetic	Macromolecular molds for the synthesis of complementary macromolecules, as in DNA REPLICATION; GENETIC TRANSCRIPTION of DNA to RNA, and GENETIC TRANSLATION of RNA into POLYPEPTIDES.	Genetic Template,Genetic Templates,Template, Genetic
D016133	Polymerase Chain Reaction	In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships.	Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain