Method for rapid amplification of DNA

a dna amplification and rapid technology, applied in the field of dna amplification, can solve the problems of limiting the usefulness of the information obtained from the dna sample, and achieve the effects of reducing the risk of sample contamination, facilitating high-throughput screening, and overcoming inherent drawbacks

Inactive Publication Date: 2006-11-02
INVITROGEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0023] The present disclosure seeks to overcome the drawbacks inherent in other methods of DNA amplification by providing a simple and direct method for amplifying DNA. The method of the present disclosure preferably amplifies DNA in a sequence-independent manner using a single reaction mixture and a single programmable thermocycling reaction. This method can be used to amplify trace amounts of DNA, including genomic DNA from small tissue or blood samples, such as fine needle aspirates or single tissue sections, or even from a single cell. The single reaction mixture used in this method also greatly reduces the risk of sample contamination and facilitates high-throughput screening, and in a preferred embodiment a single heat-stable DNA polymerase is included to amplify all DNA in the single reaction mixture. This method allows DNA to be amplified from any species or organism. It is understood that the present disclosure encompasses sequence independent amplification of DNA from any source, including but not limited to human, animal, plant, yeast, viral, eukaryotic, and prokaryotic DNA.

Problems solved by technology

Other known methods of preparing DNA samples stored on a solid medium for genetic analysis or DNA amplification are inefficient and inconsistent, thereby limiting the usefulness of the information obtained from the DNA samples.

Method used

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  • Method for rapid amplification of DNA
  • Method for rapid amplification of DNA
  • Method for rapid amplification of DNA

Examples

Experimental program
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Effect test

example 1

[0203] The efficiency of PCR™ amplification of DNA samples stored on FTA paper processed using the presently disclosed precipitation method and the commercially produced FTA Purification Reagent (manufactured by Whatman) protocol was compared. First, four bloodstain DNA samples stored on FTA paper were processed using the disclosed precipitation method according to the following protocol: A small circle (1-3 mm) in the FTA paper sample was excised by the commercial Harris Micro-Punch manufactured by Shunderson Communication, Ottawa, Ontario, Canada, and washed with distilled water. The circle was transferred to a 96-well plate and soaked in 200 μl of DD H2O for 20 minutes. The water was changed and the circle was soaked in water again for 5 minutes. The water was next replaced with 200 μl of 0.3M NaOAc / Ethanol (50 / 50 v / v) solution and the paper was soaked for 5 minutes to fix the genomic DNA on the paper. The solution was removed and the paper was washed in 200 μl of 80% ethanol for...

example 2

[0207] The method of amplifying whole genomic DNA of the present disclosure was used to amplify genomic DNA from bovine bloodstain samples stored on FTA paper. This method combines DNA extraction and amplification in a single operation by allowing genomic DNA in a bovine bloodstain sample to be amplified in a single reaction mixture using a single thermocycling reaction. Therefore, this method greatly reduces the risk of sample contamination and facilitates high-throughput screening. Additionally, 96-well or 384-well plates can be utilized for amplification of genomic DNA stored on ETA paper using the presently disclosed methods, which greatly facilitates a high-throughput operation. The utility and efficiency of the presently disclosed method of DNA amplification was tested by comparing PCR amplification of a known bovine SNP locus using DNA amplified by the disclosed method and DNA bound directly to FTA paper. In both experiments, the DNA samples stored on FTA paper were processed...

example 3

[0213] To compare the efficiency of the presently disclosed method of DNA amplification, DNA samples stored on FTA paper were first processed using the presently disclosed precipitation method or the commercial FTA Purification Reagent protocol, and then amplified according to the disclosed DNA amplification method. Two sets of punches from six different bloodstains stored on FTA paper were first treated using either the disclosed precipitation method or the FTA Purification Reagent protocol as outlined in Example 1. Next, the two sets of DNA samples were amplified according to the disclosed DNA amplification method, as outlined in Example 2.

[0214]FIG. 4. demonstrates the results of genomic DNA amplification using the disclosed method of DNA amplification. The figure compares the efficiency of the disclosed DNA amplification method when the DNA sample is processed using the FTA Purification Reagent protocol (lanes 2-7) versus the disclosed precipitation method (lanes 8-13). FIG. 4....

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Abstract

The present disclosure relates to methods of DNA amplification with a first primer that has a random sequence of nucleotides at its 3' end and a generic sequence 5' of the random nucleotides, as well as a second primer with the generic sequence of the first primer. The disclosure further relates to a method of precipitating DNA on a solid medium to improve DNA amplification. In a preferred embodiment, the presently disclosed methods are used for high-throughput genotyping of DNA samples, such as bloodstains or trace amounts of DNA.

Description

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0001] Not applicable. REFERENCE TO A “Microfiche Appendix”[0002] Not applicable. BACKGROUND OF THE INVENTION [0003] 1. Field of the Invention [0004] The present disclosure relates generally to the field of DNA amplification and more particularly to the field of amplifying any stretch of DNA in a sequence-independent manner. [0005] 2. Description of Related Art [0006] The following description includes information that may be useful in understanding the present disclosure. It is not an admission that any of the information provided herein is prior art, or relevant, to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art. [0007] It is well known that there is often an association between genetic variation and phenotype manifestation. Genetic variations and their associated phenotypes are studied using various methods of genotyping genomic DNA. A Single Nucleotide Po...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12P19/34G01N33/50C12N15/09
CPCC12Q1/6846C12Q1/6858C12Q1/686C12Q2525/179C12Q2525/155
Inventor JI, WANGREGG, KEQINREUS, BONNIEKEMPPAINEN, JONTAYLOR, KRISTENDAVIS, SCOTT
Owner INVITROGEN
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