Genome sequence tags

• US 20030186251A1
• Filed: 04/01/2002
• Published: 10/02/2003
• Est. Priority Date: 04/01/2002
• Status: Abandoned Application

• Current Assignee: Brookhaven Science Associates, LLC (Government of the United States of America)
• Original Assignee: Brookhaven Science Associates, LLC (Government of the United States of America)
• Inventors: Van der Lelie, Daniel, Dunn, John J., Krause, Maureen K.
• Application Number:
  US10/113,916
• Publication Number:
  US 20030186251A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor

Abstract

Genomic Signature Tags (GSTs) are the products of a method for identifying and quantitatively analyzing genomic DNAs. The DNA is initially fragmented with a type II restriction enzyme. An oligonucleotide adapter containing a recognition site for MmeI, a type IIS restriction enzyme, is then used to release 21 bp tags from fixed positions in the DNA relative to the sites recognized by the fragmenting enzyme. These tags are PCR-amplified, purified, concatenated into longer molecules, and then cloned and sequenced. The tag sequences and abundances are used to create a GST profile that can identify and quantify the genome of origin within any complex DNA isolate. The total number of GSTs generated from a sample is determined by the incidence of recognition sites for the initial fragmenting enzyme.

Claims

1. A method for generating a genome signature tag library, comprising:

a) providing a genomic DNA-containing sample;

b) contacting genomic DNA in the genomic DNA-containing sample with a type II restriction enzyme, under conditions appropriate for complete digestion of the genomic DNA by the type II restriction enzyme, thereby generating a plurality of digestion fragments, each digestion fragment having complementary cohesive termini;

c) incubating the digestion fragments of step b) with a molar excess of biotinylated duplex complementary adapter fragments, each having only one cohesive end, under conditions appropriate for ligating one biotinylated duplex complementary adapter fragment to each end of the digestion fragments of step b);

d) contacting the product of step c) with the restriction enzyme NlaIII, under conditions appropriate for complete digestion by the restriction enzyme;

e) recovering labelled fragments following the digestion of step d) by capture with streptavidin-coated magnetic beads;

f) incubating the magnetic beads bearing recovered labelled fragments of step e) with a molar excess of duplex linker having NlaIII cohesive termini, under conditions appropriate for ligation of complementary cohesive termini, thereby generating a recognition sequence for the restriction enzyme MmeI at any location in which the duplex linker is ligated to an NlaIII cohesive termini;

g) removing excess linkers from the incubation mixture of step f);

h) incubating the magnetic beads with bound recovered labelled fragments from step g) with the restriction enzyme MmeI under conditions appropriate for complete digestion of the ligation product of step f), thereby releasing a fragment comprising duplex linker of step f) and an appended genomic signature tag;

i) recovering the genomic signature tag-containing fragment of step h);

j) incubating the recovered fragment of step i) with a molar excess of adapter fragment, the adapter fragment having a 16-fold degenerate 31 overhang, the adapter fragment adding two consecutive T residues and a second NlaIII restriction enzyme recognition sequence following productive ligation to a free MmeI restriction enzyme-generated end, the incubation being carried out under conditions appropriate for ligation of the adapter fragment to any recovered fragment having a free MmeI restriction enzyme-generated end;

k) amplifying the ligation products of step j) with a pair of biotinylated primers specific for the duplex linker of step f) and the adapter fragment of step j);

l) incubating the amplification product of step k) with the restriction enzyme NlaIII under conditions appropriate for complete digestion of the amplification product by the restriction enzyme;

m) capturing biotinylated end fragments generated in step l) using streptavidin-coated magnetic beads leaving tag fragments, comprising 19-base pair duplex genomic signature tags with NlaIII cohesive end tags, free in solution;

n) isolating the tag fragments from step m);

o) ligating the isolated tag fragments of step n) to form concatemers;

p) isolating concatemers of sufficient minimum length;

q) cloning the concatemers of step p) in a vector; and

r) transforming prokaryotic cells with the cloned concatemers of step q) to generate a genome sequence tag library.