Efficient linking of nucleic acid segments

Details Efficient linking of nucleic acid segments Efficient linking of nucleic acid segments

1998-09-28

2001-03-20

Horlick, Kenneth R. (Department: 1656)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S091200, C435S069100, C435S069700, C435S006120

Reexamination Certificate

active

06204025

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Technical Field
This invention relates to a method for the efficient amplification of target DNA segments which is particularly advantageous for those target genes containing many small exons. Specifically, the method involves a rapid polymerase chain reaction technique for linking multiple gene segments from a single gene or multiple genes into a large DNA molecule suitable for further analysis.
2. Description of the Background Art
The technique known as the polymerase chain reaction (PCR) is a method of amplification of genomic DNA (Saiki et al., Science: 1350-1354 (1985)). Typically, the method uses two oligonucleotide primers to amplify a single DNA segment millions of times. Each cycle of DNA replication from the original primer(s) produces a product which serves as a template for further primer-dependent replication. This feature of the method results in exponential increases in the desired DNA product with each round of amplification, and a rapid accumulation of DNA. Now an automated technique performed using a thermal cycler and thermostable DNA polymerases, PCR is widely used by molecular biologists to prepare large amounts of synthetic DNA.
This revolutionary technique has been used in a wide variety of fields in molecular biology, and has made possible the rapid identification of disease-associated genes. Using the PCR, it has become feasible to diagnose inherited disorders and susceptibility to disease at the molecular level. Nevertheless, several disadvantages and limitations are recognized in the technique and its application to certain genes.
Customarily, when using PCR to amplify genomic DNA, each gene segment is amplified separately and then analyzed. When the gene of interest contains more than one exon, each exon must be amplified individually using a separate PCR, and then linked together to form a long DNA molecule representing the entire gene (Ho, et al., 1989; Horton, et al., 1989). Because no more than two gene segments can be linked together in each joining PCR, the more exons the gene of interest contains, the more separate amplifications must be individually performed and the more PCRs are needed to link the segments together. For genes which contain a lot of small exons, the preparation of a single gene for analysis can become prohibitively labor-intensive, and require a great deal of time, particularly when the small target exons must be individually scanned for mutations or polymorphisms.
It has been shown that simultaneous amplification of more than one DNA segment can be achieved with a Multiplex PCR using primers tagged with an unrelated 20 nucleotide sequence from bacteriophage M13mp18 (Shuber et al., 1995). However, with this method, products amplified with primers lacking the 20 nucleotide sequence were not reliably produced due to differences in hybridization kinetics among the primers. Using the prior art method, therefore, tagging each primer with an identical 20 nucleotide sequence is necessary to achieve efficient amplification of multiple sequences. This prior art method thus allows multiple amplifications, but the products of the amplification all contain identical unrelated sequences which would have to be removed or extended before they could be linked to form one, long DNA molecule containing all portions of the gene of interest.
After the individual gene segments have been separately amplified, further independent steps are needed to reconstruct the complete desired gene sequence from the smaller segments, with or without an introduced mutation, before the entire gene is ready for analysis. Prior art methods for linking sections of DNA using PCR involve the joining of two segments at a time, each PCR followed by a purification step. (Ho et al., 1989; Kim et al., 1996). The joining of several gene segments together, therefore, requires multiple PCRs and multiple purification steps. For example, joining four exons to form one complete gene using these prior art methods would require four separate amplifying PCRs, four separate purifications of the products and three joining PCRs. Each additional DNA segment in the gene would require an additional PCR to link it to the others, increasing both the time and expense of preparing the DNA for analysis using prior art methods.
In summary, the prior art methods of producing an amplified copy of an entire gene composed of multiple linked exons or an amplified copy of a long DNA molecule composed of gene segments from more than one gene have the disadvantage that multiple PCRs are generally required at each step. Previously, it has not been possible to efficiently amplify multiple gene segments in a single PCR to yield products that had complementary ends suitable for easy, rapid linkage in a second single PCR.
Consequently, there has been a need in the field for a simple and rapid method allowing amplification of DNA which contains several linked DNA segments which occur in non-adjacent portions of target DNA. There is a need for a method which can produce an amplified DNA molecule containing an entire gene of linked exons from genomic DNA, e.g., for DNA diagnosis.
SUMMARY OF THE INVENTION
The present invention provides a method of linking by PCR DNA segments which occur in non-adjacent portions of target DNA wherein each DNA segment contains a sequence complementary to a sequence in the DNA segment or segments to which it is to be linked, comprising using a) a first primer which is complementary to the antisense strand of the first DNA segment to be linked and a second primer which is complementary to the sense strand of the last DNA segment to be linked; and b) at least one polymerase lacking 3′→5′ exonuclease activity and at least one polymerase containing 3′→5′ exonuclease activity.
In addition, the present invention provides a method of producing and amplifying DNA containing at least three linked DNA segments which occur in non-adjacent portions of target DNA, comprising a) providing a first primer and a second primer for each DNA segment to be amplified, i) the first primer (termed the D primer) having a 3′ portion which is complementary to the 3′ end of the antisense strand of the DNA segment and a 5′ tail which is complementary to the 5′ end of the second primer for the previous DNA segment or to a sequence internal to the previous DNA segment; ii) the second primer (termed the U primer) having a 3′ portion which is complementary to the 3′ end of the sense strand of the DNA segment and a 5′ tail which is complementary to the 5′ end of the first primer for the subsequent segment or to a sequence internal to the subsequent DNA segment; b) amplifying the at least three DNA segments by multiplex PCR using the pairs of first and second primers; and c) subjecting the at least three amplified DNA segments to a linking PCR using a sense primer which is complementary to the antisense strand of the first segment to be linked and an antisense primer which is complementary to the sense strand of the last segment to be linked.


REFERENCES:
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patent: 5556772 (1996-09-01), Sorge et al.
patent: 5876940 (1999-03-01), Groden et al.
patent: 9207075 (1992-04-01), None
patent: 9516028 (1995-06-01), None
Liu, Q., “Linking PCR splices small exons into a large DNA molecule amenable to rapid mutation screening”American J. Human Genetics, vol. 61, No. 4, Oct. 1997, p. a223.

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