Methods, compositions, kits, and uses for analysis of nucleic acids comprising repeating a/t-rich segments

Abstract

Described herein are methods, compositions, kits, and uses thereof for analysis of nucleic acid segments comprising a repeating A/T-rich segment, wherein the repeating A/T-rich segment is: (i) a homopolymeric segment comprising at least 10 A residues, at least 10 T residues, or at least 10 U residues, wherein the at least 10 A, T, or U residues are consecutive or interrupted once by one to three other nucleotides; or (ii) a segment comprising (TnA)m, (ATn)m, (TAn)m, or (AnT)m, wherein n is 2 or greater and m is such that the length of the repeating A/T-rich segment is 10 or more residues.

1 - 73 . (canceled) 
     
     
         74 . A kit comprising:
 at least two primers; and   NTPs in an AT/GC ratio of 8 or higher,   
       wherein the at least two primers are capable of amplifying at least one DNA template comprising:
 (i) a homopolymeric segment of at least 10 consecutive A residues or at least 10 consecutive T residues; 
 or (ii) a repeating A/T-rich segment comprising (T n A) m , (AT n ) m , (TA n ) m , or (A n T) m , wherein n is 2 or greater and m is such that the length of the repeating NT-rich segment is 10 or more residues; 
 
       wherein the at least one DNA template further comprises target and non-target nucleic acid sequences. 
     
     
         75 . The kit of  claim 74 , wherein the at least two primers comprise at least one primer comprising a covalently attached label. 
     
     
         76 . The kit of  claim 74 , wherein the at least two primers comprise a first primer that hybridizes upstream of the rs10524523 variable length polymorphism of the TOMM40 gene and a second primer that hybridizes downstream of the rs10524523 variable length polymorphism of the TOMM40 gene. 
     
     
         77 . The kit of  claim 74 , wherein the kit further comprises at least one polymerase. 
     
     
         78 . The kit of  claim 74 , wherein the AT/GC ratio is a value in the range from about 10 to about 40. 
     
     
         79 . The kit of  claim 74 , wherein the NTPs comprise dNTPs or rNTPs, and are present in a total NTP concentration ranging from about 0.5 mM to about 5 mM. 
     
     
         80 . The kit of  claim 74 , wherein dGTP is present in a concentration ranging from about 10 μM to about 400 μM and cytidine is present in a concentration ranging from about 10 μM to about 400 μM. 
     
     
         81 . The kit of  claim 74 , wherein dTTP is present in a concentration ranging from about 700 μM to about 2000 μM and adenosine is present in a concentration ranging from about 700 μM to about 2000 μM. 
     
     
         82 . The kit of  claim 74  further comprising Mg 2+ , wherein the Mg 2+  is present in a molarity ranging from about 80% to about 150% of the molarity of total NTPs. 
     
     
         83 . The kit of  claim 74  further comprising Mg 2+ , wherein the Mg 2+  is present in a concentration ranging from about 1.5 mM to about 3 mM. 
     
     
         84 . The kit of  claim 74 , wherein the at least one nucleic acid template comprises a homopolymeric segment comprising 10 to 40 consecutive A residues or 10 to 40 consecutive T residues. 
     
     
         85 . The kit of  claim 74 , wherein the at least one nucleic acid template comprises a homopolymeric segment of at least 10 consecutive T residues. 
     
     
         86 . The kit of  claim 74 , wherein the at least one nucleic acid template comprises a segment comprising (T n A) m , (AT n ) m , (TA n ) m , or (A n T) m , wherein n is 2 or greater and m is such that the length of the repeating A/T-rich segment is 10 or more residues. 
     
     
         87 . The kit of  claim 74 , wherein at least one primer comprises a 3′-terminal sequence that specifically hybridizes to two or more consecutive A residues or to two or more consecutive T residues. 
     
     
         88 . The kit of  claim 87 , wherein the at least one primer comprises a 3′-terminal sequence comprising 4 to 9 consecutive A residues or 4 to 9 consecutive T residues. 
     
     
         89 . The kit of  claim 74 , wherein at least one primer comprises a covalently attached label. 
     
     
         90 . The kit of  claim 77 , wherein the at least one polymerase comprises a hot-start DNA polymerase. 
     
     
         91 . The kit of  claim 74 , wherein the at least one nucleic acid template comprises a homopolymeric segment comprising at least 12 consecutive A residues or at least 12 consecutive T residues. 
     
     
         92 . The kit of  claim 74  wherein the at least one nucleic acid template comprises a homopolymeric segment in a genetic locus associated with late-onset Alzheimer's disease. 
     
     
         93 . The kit of  claim 74 , wherein the at least one nucleic acid template comprises a subset of TOMM40 sequence comprising a homopolymeric segment of at least 10 consecutive T residues.