Title: Methods for producing partially digested restriction DNA fragments and for producing a partially modified PCR product

Abstract

The present invention is an improved method of making a partially modified PCR product from a DNA fragment with a polymerase chain reaction (PCR). In a standard PCR process, the DNA fragment is combined with starting deoxynucleoside triphosphates, a primer, a buffer and a DNA polymerase in a PCR mixture. The PCR mixture is then reacted in the PCR producing copies of the DNA fragment. The improvement of the present invention is adding an amount of a modifier at any step prior to completion of the PCR process thereby randomly and partially modifying the copies of the DNA fragment as a partially modified PCR product. The partially modified PCR product may then be digested with an enzyme that cuts the partially modified PCR product at unmodified sites thereby producing an array of DNA restriction fragments.

Inventors:

Wong, Kwong-Kwok ^[1]

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1. Richland, WA

Issue Date:

Sat Jan 01 00:00:00 EST 2000

Research Org.:

Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

OSTI Identifier:

872938

Patent Number(s):

6046039

Assignee:

Battelle Memorial Institute (Richland, WA)

Patent Classifications (CPCs):

C - CHEMISTRY C12 - BIOCHEMISTRY C12N - MICROORGANISMS OR ENZYMES

C - CHEMISTRY C12 - BIOCHEMISTRY C12P - FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {

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C - CHEMISTRY C12 - BIOCHEMISTRY C12N - MICROORGANISMS OR ENZYMES

C12N15/10 - Processes for the isolation, preparation or purification of DNA or RNA

C - CHEMISTRY C12 - BIOCHEMISTRY C12P - FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {

C12P19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides

C - CHEMISTRY C12 - BIOCHEMISTRY C12Q - MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS

C12Q1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q2521/301 - Endonuclease
C12Q2525/117 - incorporating modified base
C12Q2525/125 - incorporating agents resulting in resistance to degradation
C12Q2525/137 - incorporating/modifying moieties to eliminate restriction sites
C12Q2531/113 - PCR

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DOE Contract Number:  

AC06-76RL01830

Resource Type:

Patent

Country of Publication:

United States

Language:

English

Subject:

methods; producing; partially; digested; restriction; dna; fragments; modified; pcr; product; improved; method; fragment; polymerase; chain; reaction; standard; process; combined; starting; deoxynucleoside; triphosphates; primer; buffer; mixture; reacted; copies; improvement; adding; amount; modifier; step; prior; completion; randomly; modifying; enzyme; cuts; unmodified; sites; array; pcr product; partially modified; modified pcr; chain reaction; dna polymerase; improved method; dna fragments; polymerase chain; dna fragment; restriction fragments; restriction fragment; nucleoside triphosphate; /435/436/999/