US2008233566A1PendingUtilityA1

Methods and materials for detecting mutations in quasispecies having length polymorphisms

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Assignee: BAYER HEALTHCARE LLCPriority: Aug 20, 2004Filed: Aug 19, 2005Published: Sep 25, 2008
Est. expiryAug 20, 2024(expired)· nominal 20-yr term from priority
Inventors:Arejas UzgirisSharon Kemp
Y10S435/975C12Q 1/6869C12Q 2600/156C12Q 1/703
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Claims

Abstract

The present invention is directed to a method for detecting the presence or absence of a mutation of interest in the nucleic acid of a pathogen, wherein the mutation of interest is located adjacent to a length polymorphism defining multiple quasispecies of the pathogen.

Claims

exact text as granted — not AI-modified
1 . A primer combination comprising a set of bi-directional sequencing primers encompassing a region comprising the HR1 and HR2 domains of HIV-1, wherein the primer combination comprises:
 (a) a forward primer selected from the group consisting of one or more of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO:14, and fragments thereof of 15 or more nucleotides; and   (b) a reverse primer selected from the group consisting of one or more of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and fragments thereof of 15 or more nucleotides.   
     
     
         2 . A primer combination according to  claim 1 , wherein the primer combination comprises a set of bi-directional sequencing primers encompassing a region consisting essentially of nucleotides 7329-7614 of SEQ ID NO:1 or fragments thereof of 15 or more nucleotides. 
     
     
         3 . The primer combination according to  claim 2 , wherein the set of bi-directional sequencing primers encompasses a region consisting essentially of nucleotides 7388-7549 of SEQ ID NO:1 or fragments thereof of 15 or more nucleotides. 
     
     
         4 . The primer combination according to  claim 3 , wherein the set of bi-directional sequencing primers encompasses a region consisting essentially of nucleotides 7492-7522 of SEQ ID NO:1 or fragments thereof of 15 or more nucleotides. 
     
     
         5 . The primer combination according to  claim 4 , wherein the set of bi-directional primary sequencing primers comprises two or more primers selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and fragments thereof of 15 or more nucleotides. 
     
     
         6 . The primer combination of  claim 5 , wherein the set of bi-directional sequencing primers comprises:
 (a) at least one forward primer selected from the group consisting of SEQ ID NO:31 and SEQ ID NO:32, and fragments thereof of 15 or more nucleotides, and   (b) at least one reverse primer selected from the group consisting of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and fragments thereof of 15 or more nucleotides.   
     
     
         7 . A method for detecting the presence or absence of a mutation of interest in the nucleic acid of a pathogen, wherein the mutation of interest is located adjacent to a length polymorphism defining multiple quasispecies of the pathogen, comprising:
 a) obtaining from the patient sample a double-stranded DNA template encompassing the mutation of interest;   b) sequencing a first strand of a region of the DNA template containing the mutation of interest;   c) sequencing a second strand of a region of the DNA template containing the mutation of interest, wherein the region of the DNA template sequenced that is common to the first strand and second strand excludes the length polymorphism; and   d) comparing the sequence of the first strand with the sequence of the second strand to obtain complementary strand confirmation of the sequence of the mutation of interest.   
     
     
         8 . A method according to  claim 7 , wherein the pathogen is HIV-1. 
     
     
         9 . The method according to  claim 8 , wherein the mutation of interest is located in the HIV-1 envelope glycoprotein gp41. 
     
     
         10 . The method of  claim 9 , wherein the mutation of interest is located within position 36-45 of gp41. 
     
     
         11 . The method according to  claim 7 , wherein the region of the DNA template sequenced that is common to the first strand and second strand and excludes the length polymorphism consisting essentially of nucleotides 7329-7614 of SEQ ID NO:1 or fragments thereof of 15 or more nucleotides. 
     
     
         12 . The method according to  claim 7 , wherein the region of the DNA template sequenced that is common to the first strand and second strand and excludes the length polymorphism consisting essentially of nucleotides 7388-7549 of SEQ ID NO:1 or fragments thereof of 15 or more nucleotides. 
     
     
         13 . The method according to  claim 7 , wherein the region of the DNA template sequenced that is common to the first strand and second strand and excludes the length polymorphism consisting essentially of nucleotides 7492-7522 of SEQ ID NO:1 or fragments thereof of 15 or more nucleotides. 
     
     
         14 . The method according to  claim 7 , wherein the sequence is determined using primers selected from the group consisting of one or more primers selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and fragments thereof of 15 or more nucleotides. 
     
     
         15 . The method according to  claim 7 , wherein the sequence is determined using forward primers comprising one or more primers selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, and fragments thereof of 15 or more nucleotides, and reverse primers comprising one or more primers selected from the group consisting of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and fragments thereof of 15 or more nucleotides. 
     
     
         16 . The method according to  claim 7 , wherein the sequence is determined using primers selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and fragments thereof of 15 or more nucleotides. 
     
     
         17 . The method according to  claim 16 , wherein the sequence is determined using a forward primer selected from the group consisting of one or more of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and fragments thereof of 15 or more nucleotides. 
     
     
         18 . The method according to  claim 16 , wherein the primer is a reverse primer selected from the group consisting of one or more of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and fragments thereof of 15 or more nucleotides. 
     
     
         19 . The method according to  claim 16 , comprising a primer combination comprising a set of bi-directional sequencing primers encompassing a region encompassing the HR1 and HR2 domains of HIV-1, wherein the primer combination comprises:
 (a) a forward primer selected from the group consisting of one or more of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and fragments thereof of 15 or more nucleotides; and   (b) a reverse primer selected from the group consisting of one or more of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and fragments thereof of 15 or more nucleotides.   
     
     
         20 . A kit for detecting the presence or absence of a mutation of interest in a pathogen in a sample containing multiple quasispecies of the pathogen having mixed length polymorphisms, wherein the mutation of interest is located adjacent to the length polymorphism, comprising a first primer for sequencing a first strand of a region of a DNA template containing the mutation of interest and a second primer for sequencing a second strand of a region of the DNA template containing the mutation of interest, wherein the region defined by the first primer and second primer excludes the length polymorphism. 
     
     
         21 . A kit according to  claim 20 , wherein the kit comprises one or more an oligonucleotide primers selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and fragments thereof of 15 or more nucleotides. 
     
     
         22 . A kit according to  claim 21 , wherein the kit comprises one or more an oligonucleotide primers selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and fragments thereof of 15 or more nucleotides.

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