Lead Inventors:
Maria DeFatima Bonaldo, Ph.D.,
Marcelo Bento Soares Ph.D.
Normalized cDNA library capable of conversion into single-stranded circles and producing complementary nucleic acid molecule:
Due to the difficulty in identifying rare mRNA transcripts represented in cDNA libraries, normalization has been proposed as a way to increase the frequency of the least prevalent cDNAs and to reduce the frequency of the most abundant clones. However, approaches of normalization based on annealing kinetics suffer from at least two limitations: the restriction of including only 3' coding fragments in the normalized library, and the need for cDNAs to be relatively short and homogeneous in length.
Normalized cDNA library capable of conversion into single-stranded circles and producing complementary nucleic acid molecules:
This technology consists of a new method for normalizing a cDNA library constructed in a vector capable of being converted into single-stranded circles and producing complementary nucleic acid molecules to the single-stranded circles.
The process of normalization involves:
• converting the cDNA library into single-stranded circles
• generating anti-sense RNA in vitro to the single-stranded circles
• hybridizing the single-stranded circles with the antisense RNA to produce partial duplexes to an appropriate C
ot, and
• purifying the unhybridized single-stranded circles from the hybridized single-stranded circles
This procedure for normalizing cDNA libraries does not impose any length constraints on the cDNAs, as no cycle of cDNA amplification by polymerase chain reaction is needed. In addition, the 5' coding regions are included in the normalized library because the library is directionally cloned, therefore allowing the 3' end of the clone to contain the 3' terminal exon of the mRNA with a short polyadenylation track and a recognizable polyadenylation signal sequence at the appropriate position.
Applications:
• Creating normalized cDNA libraries
Advantages:
• No restriction on length and homogeneity of cDNAs
• Ability to include 5' coding regions in normalized cDNA library
Patent Status: Patent Issued (US 5,702,898) ~ see link below.
Licensing Status: Available for Licensing