Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-11-03
2001-02-13
Horlick, Kenneth R. (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S183000, C435S283100, C435S287100, C536S023100, C536S023200, C536S023500, C536S024300, C536S024310, C536S025320
Reexamination Certificate
active
06187544
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a simple, rapid, and cost effective method for the identification and isolation of full length cDNA clones. The method also provides a means to isolate two or more separate, full length cDNA clones for each target gene sequence of interest. This method provides an alternative to traditional library screening technologies.
BACKGROUND OF THE INVENTION
Identification, sequencing and characterization of genes is a major goal of modern scientific research. By identifying genes, determining their sequences and characterization of their biological function, it is possible to employ recombinant technology to produce large quantities of valuable gene products, e.g. proteins and peptides. Additionally, knowledge of gene sequences can provide a key to diagnosis, prognosis and treatment in a variety of disease states in plants and animals which are characterized by inappropriate expression and/or repression of selected genes or by the influence of external factors, e.g., carcinogens or teratogens, on gene function.
As thousands of EST (expressed sequence tag) assemblies for potentially therapeutic gene targets are present in both public and private sequence databses. Analysis of assembly databases can provide insight as to which genes should be further studied for potential use as therapeutic targets or agents. However, such studies are limited unless the intact full length sequence is available for use. Advances in DNA sequencing technology and computational methodologies have drastically altered the rate at which sequencing projects and gene identification can proceed. Literally thousands of cDNA clones, or ESTs, can be randomly sequenced weekly and then computationally assembled into distinct genes. As roughly only 10% of the members of a standard, polyA primed cDNA library are full length, these computational assemblies rarely contain the sequence of the entire expressed gene. This necessitates several rounds of library screening in order to identify an intact full length cDNA clone for practically any gene one wishes to study. These screening procedures can often be inefficient, costly, and time consuming.
Accordingly, there exists a need for a more efficient and rapid method of identifying and isolating thousands of full length cDNA clones. This method must be simple, robust, and enable the identification of multiple cDNA clones for the target gene of interest.
SUMMARY OF THE INVENTION
The present invention provides a method for high throughput cloning of full length cDNA sequences. This method uses a plurality of clone arrays prepared from cDNA libraries which have been preferably enriched for 5′ mRNA sequences and size fractionated into several discrete ranges (sub-libraries). These arrays are used to rapidly identify the full length cDNA sequence for a DNA segment of interest.
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Bergsma Derk Jon
Mooney Jeffrey L
Han William T.
Horlick Kenneth R.
King William T.
Kinzig Charles M.
Siew Jeffrey
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