DNA template tailoring using PNA and modified nucleotides

摘要

Disclosed is a method whereby a repetitive nucleic acid sequence, such as a short tandem repeat (STR), may be characterized as to its length. Pyrosequencing is used to sequence an STR repetitive region to measure the length of STRs in a rapid manner. A combinatorial approach is disclosed for the addition of multiple nucleotides (e.g., two mononucleotides) at a time by the polymerase, which reduces the sample analysis time by half. In addition, modified nucleic acids, such as peptide nucleic acids, are used as blocking probe to stop polymerization on the flanking region which makes it possible to use pyrosequencing for DNA length measurement both in the case of homozygous or heterozygous samples for varying repeat patterns of different markers. Further, dideoxynucleotides are added to stop polymerization in the flanking region of the STR.

主权项

1. A method for characterizing by length of a repetitive sequence in a sample nucleic acid, said repetitive sequence having a location between a 5′ flanking region and a 3′ flanking region, the method comprising the steps of:
(a) hybridizing a polymerization primer to said 3′ flanking region; (b) hybridizing a modified oligonucleotide to said 5′ flanking region, said modified oligonucleotide being resistant to displacement by a polymerase enzyme; (c) extending the polymerization primer in a 5′ to 3′ direction by using said polymerase enzyme, said extending stopping at 5′ end of the modified oligonucleotide; and (d) said extending comprising the step of adding nucleotides complementary to the sample sequence and the step of adding a dideoxy nucleotide selected to be only complementary to a nucleotide in the 5′ flanking region, whereby extending proceeds from said polymerization primer to the modified oligonucleotide unless terminated by an added dideoxynucleotide, wherein the repetitive sequence is characterized by length of extending in step (c) and (d).