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US 2023/0399693 A1Published application
Targeted sequencing method and kit thereof for detecting gene alteration
AssigneeACT Genomics (IP) Co., Ltd.
InventorsYi-Hsuan LAI, Yu-Ling CHEN, An HSU, Pei-Yi LIN, Hua-Chien CHEN, Shu-Jen CHEN
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Abstract
The present application provides a method for determining a gene alteration, such as a gene fusion. The present application also provides a kit for determining a gene alteration. The present application further provides a method for treating a subject by determining whether a subject is at risk for a particular cancer type or genotype and administering proper treatment.
Claims28
A method for detecting a gene alteration in a biological sample, comprising steps of:
- (a) mixing a target RNA obtained from the biological sample, a template-switching oligo, a reverse transcriptase, and a reverse transcription (RT) primer in a mixture, wherein the template switch oligo comprises a first universal primer sequence and the RT primer comprises a second universal primer sequence;
- (b) subjecting the mixture to a condition under which reverse transcription occurs to provide a target DNA complementary to the target RNA,
- (c) amplifying the target DNA with a first pair of primers to obtain a first PCR product, wherein the first set of primers comprise a gene specific primer and a universal primer, the gene specific primer comprises a 5′-end first or second adapter sequence and a 3′-end sequence that hybridizes to a target gene under stringent conditions, the universal primer comprises a 5′-end first or second adapter sequence and a 3′-end sequence comprising the first or second universal primer sequence, and the first and second adapter sequences are different from each other;
- (d) amplifying the first PCR product with a second pair of primers to obtain a second PCR product, wherein each of the second pair of primers comprises a 5′-end third or fourth adapter sequence and a 3′-end sequence that hybridizes to the first or second adaptor sequence, respectively, and the third and fourth adapter sequences are different from each other;
- and
- (e) analyzing the second PCR product to detect the presence of the gene alteration.
3 more elements
A kit for detecting a gene alteration in a biological sample, comprising:
- (a) a template switch oligo comprising a first universal primer;
- (b) a reverse transcription (RT) primer comprising a stem-loop structure and an overhang structure of at least 5 random tail nucleotides, wherein the RT primer comprises a second universal primer;
- (c) one or more gene specific primers each comprising a first or second adapter sequence and universal primers each comprising a first or second adapter sequence, respectively, wherein the first adapter sequence of the gene specific primer is different from the second adapter sequence of the universal primer or the second adapter sequence of the gene specific primer is different from the first adapter sequence of the universal primer;
- (d) a primer pair complementing the first and second adapters, respectively, wherein each primer of the primer pair comprises a third or fourth adapter sequence, respectively, and wherein the third and fourth adapter sequences are different from each other;
- (e) a reverse transcriptase;
- (f) a DNA polymerase;
- and
- (g) deoxy-ribonucleoside triphosphate (dNTP).
5 more elements
A kit for detecting a gene alteration in a biological sample, comprising:
- (a) a template switch oligo comprising a first universal primer;
- (b) a reverse transcription (RT) primer comprising a linear structure of at least 5 random tail nucleotides, wherein the RT primer comprises a second universal primer;
- (c) one or more gene specific primers each comprising a first or second adapter sequence and universal primers each comprising a first or second adapter sequence, respectively, wherein the first adapter sequence of the gene specific primer is different from the second adapter sequence of the universal primer or the second adapter sequence of the gene specific primer is different from the first adapter sequence of the universal primer;
- (d) a primer pair complementing the first and second adapters, respectively, wherein each primer of the primer pair comprises a third or fourth adapter sequence, respectively, and wherein the third and fourth adapter sequences are different from each other;
- (e) a reverse transcriptase;
- (f) a DNA polymerase;
- and
- (g) deoxy-ribonucleoside triphosphate (dNTP).
5 more elements
Dependencies are inferred from the claim text.