Linked Life Data

7828884

Source:http://linkedlifedata.com/resource/pubmed/id/7828884

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1-2
pubmed:dateCreated
1995-2-22
pubmed:databankReference
pubmed:abstractText
In order to obtain the 5' ends of the three mouse calmodulin (CaM) cDNAs, we modified the standard 5' RACE (rapid amplification of cDNA ends) method to use degenerate synthetic oligodeoxyribonucleotides to prime cDNA synthesis of all three CaM mRNAs. In this modified method, the degenerate primers were annealed to mRNAs in an incubation step prior to the reverse transcription reaction. Separating the annealing step from the reverse transcription reaction allowed for greater stringency by using higher temperatures than could be tolerated if the reverse transcriptase were present. Annealing was also done with lower primer concentration and was driven by a longer incubation time. After the annealing step, cDNA synthesis was initiated by diluting the annealing mixture into a 42 degrees C buffer with reverse transcriptase. The synthesized cDNA was poly(dA)-tailed to allow PCR amplification of the first-strand cDNA with an anchor-dT17 primer and the degenerate primers. The CaM cDNAs were evident after this PCR. A second PCR, with nested gene-specific primers, was used to isolate the individual CaM cDNAs from the products of the first PCR. Three distinct CaM cDNAs were cloned and sequenced. By comparison of the 5' untranslated sequences between the mouse CaM DNAs and rat CaM cDNAs, the corresponding homologs were assigned. The results suggest that application of this modified RACE method could improve the success of isolating specific cDNAs in cases where use of a nested primer is not possible or when amino-acid sequence information is available and only degenerate primers can be designed for cloning cDNAs by the 5'-RACE method.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
0378-1119
pubmed:author
pubmed:issnType
Print
pubmed:day
30
pubmed:volume
151
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
247-51
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1994
pubmed:articleTitle
Three different calmodulin-encoding cDNAs isolated by a modified 5'-RACE using degenerate oligodeoxyribonucleotides.
pubmed:affiliation
Department of Biochemistry and Anaerobic Microbiology, Virginia Tech., Blacksburg 24061.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, Non-U.S. Gov't