Methods and materials for restriction endonuclease applications
First Claim
1. A method for labeling DNA, the method comprising the stepsa) digesting an aliquot of template DNA with a restriction endonuclease reagent that is CviJ I or Taq I and Hpa II in combination, under conditions that generate sequence-specific DNA fragments from about 20 to about 200 base pairs in length and having an average length of about 20 to about 60 base pairs;
- b) mixing an aliquot of undigested template DNA with said sequence-specific DNA fragments, denaturing said mixture of template DNA and sequence-specific DNA fragments thereby generating denatured template DNA and oligonucleotide primers;
c) annealing said primers to said denatured undigested template DNA to form a DNA-primer complex;
d) performing an extension reaction from said primers in said DNA-primer complex using a DNA polymerase in the presence of one or more nucleotide triphosphates that comprise at least one labeled nucleotide triphosphate.
0 Assignments
0 Petitions
Accused Products
Abstract
The present invention is directed to materials and methods for the quasi-random and complete fragmentation of DNA using restriction endonuclease reagents capable of cutting DNA at a dinucleotide sequence. The invention is also directed to methods for labeling DNA using template-specific oligonucleotides, for shotgun cloning, for sequencing of DNA, for epitope mapping and for anonymous primer cloning, all using fragments of DNA generated by the method of the present invention.
-
Citations
29 Claims
-
1. A method for labeling DNA, the method comprising the steps
a) digesting an aliquot of template DNA with a restriction endonuclease reagent that is CviJ I or Taq I and Hpa II in combination, under conditions that generate sequence-specific DNA fragments from about 20 to about 200 base pairs in length and having an average length of about 20 to about 60 base pairs; -
b) mixing an aliquot of undigested template DNA with said sequence-specific DNA fragments, denaturing said mixture of template DNA and sequence-specific DNA fragments thereby generating denatured template DNA and oligonucleotide primers; c) annealing said primers to said denatured undigested template DNA to form a DNA-primer complex; d) performing an extension reaction from said primers in said DNA-primer complex using a DNA polymerase in the presence of one or more nucleotide triphosphates that comprise at least one labeled nucleotide triphosphate. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
-
-
9. A kit for labeling DNA, said kit comprising in association:
-
a) a restriction endonuclease reagent comprising CviJ I or Taq I and Hpa II in combination; b) a restriction endonuclease buffer that when combined with said restriction endonuclease reagent causes said restriction endonuclease reagent to digest an aliquot of template DNA to produce sequence-specific DNA fragments from about 20 to about 200 base pairs in length and having an average length of about 20 to about 60 base pairs; and c) a labeling buffer. - View Dependent Claims (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
-
-
21. A method for thermal-cycle labeling DNA comprising the steps of:
-
a) digesting an aliquot of template DNA with a restriction endonuclease reagent under conditions wherein said template DNA is cleaved at one or more nucleotide sequences selected from the group consisting of PyGCPy, PuGCPy, PuGCPu, and PyCGPu and wherein Pu=purine and Py=pyrimidine, thereby generating sequence specific DNA fragments; b) mixing an aliquot of undigested template DNA with an excess of said sequence specific DNA fragments, denaturing said mixture of template DNA and said excess of sequence specific DNA fragments, thereby generating denatured template DNA and excess oligonucleotide primers; c) annealing said primers to said undigested template DNA to form a DNA-primer complex; d) performing an extension reaction from said primers in said DNA-primer complex using a DNA polymerase in the presence of one or more nucleotide triphosphates that comprise at least one labeled nucleotide triphosphate, thereby producing labeled extension products; e) heat-denaturing said labeled extension products from said template DNA; f) reannealing said excess primers with said template DNA and with said extension products; and g) performing at least one additional extension reaction using a DNA polymerase. - View Dependent Claims (22, 23, 24, 25, 26, 27, 28, 29)
-
Specification