Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1998-07-17
2000-09-05
McKelvey, Terry
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C12P 1934
Patent
active
061141542
ABSTRACT:
A method of direct construction and isolation of full-length target cDNA molecules obtains a large number of full-length target cDNA clones with minimum screening. This new method saves time and resources that are needed in traditional methods requiring large screenings of clones that do not contain the desired sequence. A characteristic of this method includes using a total RNA population to purify mRNA for synthesizing a mixed population of first strand cDNA. Another characteristic is the use of a short sequence of the target sequence as a forward primer for the generation of the second strand cDNAs and then separating out all non-hybridizing single stranded cDNAs. Only the double stranded cDNAs are then cloned and sequenced. In this manner, only target cDNA clones are obtained and a large amount of screening is avoided. Another characteristic is the use of directed expression of the cDNA during cloning by modifying each of the two ends of the cDNA with a different restriction enzyme, each end of the cDNA then being distinct and incompatible with the other end. The directional target cDNA is then ligated into a vector compatible with the ends of the directional cDNA. The vector-ligated cDNA is transformed into a bacterial host cell. The sequence of the isolated full-length target cDNA molecule is confirmed by sequencing.
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