Methods for determining genetic haplotypes and DNA mapping
AssigneesThe Board of Trustees of the University of Illinois, The Regents of the University of California
InventorsMing Xiao, Pui-Yan Kwok, Paul Selvin, Mathew Gordon
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Abstract
Improved methods of genetic haplotyping and DNA sequencing and mapping, including methods for making amplified ssDNA, methods for allele determination, and a DNA barcoding strategy based on direct imaging of individual DNA molecules and localization of multiple sequence motifs or polymorphic sites on a single DNA molecule.
Claims5
A method for making long (2-20 Kb) amplified ssDNA, the method comprising the steps of:
- a) amplifying by long range PCR template DNA using a first primer having a 5′ detectable label and a second primer having a 5′ phosphore to generate amplified first strands incorporating the 5′ detectable label, and amplified second strands incorporating the 5′ phosphore;
- and
- b) selectively digesting the second strands with a 5′-3′ exonuclease to provide long amplified, single-stranded first strands, wherein the 5′ detectable label inhibits digestion of the first strand by the exonuclease.
A method for allele specific labeling of multiple SNPs simultaneously for molecular haplotyping, the method comprising the steps of:
- a) contacting simultaneously a long (2-20 Kb) ssDNA template comprising a plurality of SNPs with a corresponding plurality of first and second allele specific primers corresponding to alternative alleles at the SNPs and comprising alternate fluorescent labels, under conditions wherein one of each of the first and second allele-specific primers hybridizes at each corresponding SNP;
- b) extending simultaneously with polymerase the hybridized primers to convert the ssDNA to dsDNA comprising incorporated labels of the hybridized primers;
- c) linearizing the dsDNA;
- and
- d) reading the order of the incorporated labels of the dsDNA to determine the molecular haplotype of the dsDNA.
A method for allele specific labeling of multiple SNPs sequentially for allele determination, the method comprising the steps of:
- a) contacting sequentially a long (2-20 Kb) ssDNA template comprising a plurality of SNPs and corresponding 5′ flanking sequences, with a corresponding plurality of 5′ flanking sequence-specific primers, under conditions wherein the primers hybridize at the corresponding 5′ flanking sequences;
- b) extending sequentially the 3′ end of the primers with single nucleotides corresponding to the alternative alleles at the SNP and comprising corresponding fluorescent labels, wherein the nucleotides are added by single-base primer extension or oligo ligation;
- and
- c) detecting sequentially the incorporated labels at each SNP to infer the allele of each SNP.
A method for sequencing a long (2-20 Kb) ssDNA, the method comprising the steps of:
- a) hybridizing one or more primers to a long template ssDNA confined in two-dimensions in a channel or on a solid surface;
- b) extending the 3′ end of the primers with fluorescently-labeled nucleotides by polymerase extension or fluorescently-labeled oligos by ligase ligation, to obtain fluorescently-labeled dsDNA;
- and
- c) sequentially reading each of the incorporated fluorescently-labeled nucleotides or fluorescently-labeled oligo sequence of the dsDNA using TIRF microscopy to obtain a base-by-base sequence of the dsDNA.
A method of DNA mapping, the method comprising the steps of:
- a) nicking one strand of a long (>2 Kb) double stranded genomic DNA molecule with a nicking endonuclease to introduce nicks at specific sequence motifs;
- b) incorporating fluorescent dye-labeled nucleotides at the nicks with a DNA polymerase;
- c) staining the backbone of the genomic DNA;
- d) stretching the labeled DNA molecule into linear form on a modified glass surface;
- and
- e) determining the positions of the fluorescent labels with respect to the DNA backbone using total internal reflection fluorescence (TIRF) microscopy to obtain a map or barcode of the DNA.
Dependencies are inferred from the claim text.