Method for rapid amplification of DNA

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...

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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.

[0024] The present disclosure also offers an improved method for processing DNA samples on a solid medium. 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. The method of the present disclosure seeks to overcome the drawbacks inherent in these other methods by greatly simplifying the preparation of DNA samples and improving the DNA for subsequent analysis. The method of the present disclosure precipitates the DNA sample on a solid medium using methods well known to those of skill in the art. In a preferred embodiment, the DNA sample is a bloodstain on a solid medium. The DNA processed according to the presently disclosed method can be subsequently subjected to DNA amplification using the presently disclosed methods and / or genetic analysis. This disclosed precipitation method produces more consistent results, reduces the cost of high-throughput operations, and improves the quality of DNA amplified from the DNA sample.

[0050] The DNA amplification methods of the present disclosure will be useful for amplifying small amounts of DNA, which will allow multiple sites in the DNA sample to be genotyped for high-throughput screening. Additionally, the present method will allow for the rapid construction of band specific painting probes for any chromosomal region, and can also be used to microdissect and amplify unidentifiable chromosomal regions or marker chromosomes in abnormal karyotypes. The presently disclosed method will also allow for the rapid cloning of amplified DNA for sequencing or generating DNA libraries. Thus, the method will not only be a valuable tool for genotype analysis and high-throughput screening, it should also be a valuable tool in cytogenetic diagnosis.

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.

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