Method for DNA breakpoint analysis,申请号US200812130049-传众专利搜索

Method for DNA breakpoint analysis

申请公布号：US9145587(B2)

申请号：US200812130049

申请日期：2008.05.30

申请公布日期：2015.09.29

申请人：

MONOQUANT PTY LTD

发明人：Morley Alexander Alan

分类号：C12P19/34;C12Q1/68

主分类号：C12P19/34

代理机构：

Knobbe, Martens, Olson & Bear LLP

代理人：Knobbe, Martens, Olson & Bear LLP

地址：Adelaide AU

摘要：The present invention relates to a method for identifying a DNA breakpoint and agents for use therein. More particularly, the present invention provides a method for identifying a gene translocation breakpoint based on the application of a novel multiplex DNA amplification technique. The method of the present invention facilitates not only the identification of the breakpoint position but, further, enables the isolation of the DNA segment across which the breakpoint occurs. This provides a valuable opportunity to conduct further analysis of the breakpoint region, such as to sequence across this region. The method of the present invention is useful in a range of applications including, but not limited to, providing a routine means to characterize the gene breakpoint associated with disease onset in a patient and thereby enable the design of patient specific probes and primers for ongoing monitoring of the subject disease condition. In addition to monitoring the progression of a condition characterized by the existence of the breakpoint, there is also enabled assessment of the effectiveness of existing therapeutic drugs and/or new therapeutic drugs and, to the extent that the condition is a neoplasm, prediction of the likelihood of a subject's relapse from a remissive state.

主权项：1. A method of identifying a gene breakpoint, said method comprising: (i) in a first round amplification reaction, contacting a DNA sample with: (a) one or more forward primers directed to the antisense strand of a genomic DNA region of the flanking gene or fragment thereof, said region being located 5′ relative to the gene breakpoint; and(b) one or more reverse primers directed to the sense strand of a genomic DNA region of the flanking gene or fragment thereof, said region being located 3′ relative to the gene breakpoint; wherein all of the forward primers or all of the reverse primers or all of both the forward and reverse primers are operably linked at their 5′ end to an oligonucleotide tag; and if the forward primers are operably linked to an oligonucleotide tag then the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (i)(a);if the reverse primers are operably linked to an oligonucleotide tag then the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (i)(b);if both the forward primers and the reverse primers are operably linked to an oligonucleotide tag then forward primer oligonucleotide tags are different relative to the reverse primer tags; and(c) if a forward primer tag is present then hybridizing a primer directed to the forward primer oligonucleotide tag of step (i)(a); and(d) if a reverse primer tag is present then hybridizing a primer directed to the reverse primer oligonucleotide tag of step (i)(b); (ii) amplifying the DNA sample of step (i); (iii) in a second round amplification reaction, contacting the amplicon generated in step (ii) with: (a) one or more forward primers directed to the antisense strand of a genomic DNA region of the flanking gene or fragment thereof, said region being located 5′ relative to the gene breakpoint and 3′ relative to one or more of the regions of step (i)(a); and(b) one or more reverse primers directed to the sense strand of a genomic DNA region of the flanking gene or fragment thereof, said region being located 3′ to the gene breakpoint and 5′ relative to one or more of the regions of step (i)(b); wherein all of the forward primers or all of the reverse primers or all of both the forward and reverse primers are operably linked at their 5′ end to an oligonucleotide tag; and if the forward primers are operably linked to an oligonucleotide tag then the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (iii)(a);if the reverse primers are operably linked to an oligonucleotide tag then the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (iii)(b);if both the forward primers and the reverse primers are operably linked to an oligonucleotide tag then forward primer oligonucleotide tags are different relative to the reverse primer tags and which forward and reverse primer tags of step (iii) are different relative to the forward and reverse primer tags of step (i); and(c) if a forward primer tag is present then hybridizing a primer directed to the forward primer oligonucleotide tag of step (iii)(a); and(d) if a reverse primer tag is present then hybridizing a primer directed to the reverse primer oligonucleotide tag of step (iii)(b); (iv) amplifying the DNA sample of step (iii); and (v) analysing the amplified DNA.