Genome selection and conversion method
View Patent ↗The present disclosure provides a method and a kit for selecting and enriching target sequences specific for one or more genomic regions of interest or a subset of a transcriptome using a target-capturing library of probes. The target-enriched library is generated from a random pool of deoxyribonucleic acid (DNA) fragments. The present disclosure provides an efficient and cost-effective method of target selection for targeted genome sequencing, targeted nucleic acid library creation and gene expression studies.
1. A method for enriching for target nucleic acid sequences from a mixed population of nucleic acid fragments using a blocking RNA, a single labeled target isolation probe and two adaptor molecules, the method comprising:
(a) obtaining a population of nucleic acid fragments;
(b) adding to the population of nucleic acid fragments, the blocking RNA that hybridizes to nontarget repetitive sequences;
(c) denaturing double stranded nucleic acid into single stranded nucleic acids and permitting the blocking RNA to hybridize to the single stranded nucleic acids to form RNA blocked nucleic acid;
(d) hybridizing to the target nucleic acid sequence contained in the RNA blocked nucleic acid, the labeled target isolation nucleic acid probe, wherein the label is positioned between the 5′ end and the 3′ end of the labeled probe;
(e) immobilizing the hybridized nucleic acid of (d) by means of the label and removing unbound material;
(f) removing from the target nucleic acid sequences, non target nucleic acid sequences at the 3′ end and the 5′ end of the nucleic acid fragments of (e) by means of a 5′ single strand specific exonuclease and a 3′ single strand specific exonuclease or a single 3′, 5′ single strand specific exonuclease; and
(g) ligating a 5′ adaptor nucleic acid to the 3′ end of the target nucleic acid sequence and a 3′ adaptor nucleic acid to the 5′ end of the target molecule either in parallel or sequentially and amplifying the target nucleic acid sequence therebetween.