Target DNA amplification by MIPC and PCR

Details Target DNA amplification by MIPC and PCR Target DNA amplification by MIPC and PCR

1999-05-13

2001-04-17

Horlick, Kenneth R. (Department: 1656)

Chemistry: molecular biology and microbiology

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

Preparing compound containing saccharide radical

C435S006120, C536S022100, C536S025400, C210S635000, C210S198200

Reexamination Certificate

active

06218153

ABSTRACT:

FIELD OF THE INVENTION
The present invention concerns a chromatographic method for detection of mutations in nucleic acids.
BACKGROUND OF THE INVENTION
The early diagnosis of certain diseases, especially cancer, can save many lives. In the case of cancer, and other diseases of genetic origin, early detection often depends on the availability of an appropriate analytical method which can accurately and reliably detect a putative mutation in DNA samples. This problem is exacerbated by the fact that such samples generally contain a very small population of cells containing mutant DNA in the presence of a very large predominantly normal cell population containing wild type DNA. Any separation technique which is capable of detecting mutant DNA in the presence of wild type would fail under these circumstances because the concentration of mutant DNA is simply too low to be detected relative to wild type. That is to say, the concentration of mutant DNA may be too low to detect in absolute terms. Alternatively, the concentration of mutant DNA may be sufficient to detect, but will be completely obscured because of the very large relative amount of wild type in the sample.
Increasing the amount of mutant DNA by PCR amplification of the sample would not solve the problem described above. The mutant and wild type DNA in the sample are very similar. In fact, their sequence may differ by only a single base pair. Therefore, the primers which would be used to amplify the mutant DNA would also amplify the wild type since both are present in the sample. As a result, the relative amounts of mutant and wild type DNA would not change.
Following radiation or chemotherapy, cancer patients are monitored for the presence of residual cancer cells to determine whether the patients are in remission. The effectiveness of these treatments can be monitored if small levels of residual cancer cells could be detected in a predominantly large wild type population. Traditionally, the remission status is assessed by a pathologist who conducts histological examination of tissues samples. However, these visual methods are largely qualitative, time-consuming, and costly. At best, the sensitivity of these methods permits detection of about 1 cancerous cell in 100 cells.
Analysis of DNA samples has historically been done using gel electrophoresis. Capillary electrophoresis has also been used to separate and analyze mixtures of DNA. However, these methods cannot distinguish point mutations from homoduplexes having the same base pair length.
Gel based analytical methods, such as denaturing gradient gel electrophoresis and denaturing gradient gel capillary electrophoresis, can detect mutations in heteroduplex DNA strands under “partially denaturing” conditions. The term “partially denaturing” means the separation of a mismatched base pair (caused by temperature, pH, solvent, or other known factors) in a DNA double strand while the remainder of the double strand remains intact. However, these gel based techniques are operationally difficult to implement and require highly skilled personnel. In addition, the analyses are lengthy and require a great deal of set-up time. A denaturing capillary gel electrophoresis analysis of a 90 base pair fragment takes more than 30 minutes and a denaturing gel electrophoresis analysis may take 5 hours or more. The long analysis time of the gel methodology is further exacerbated by the fact that the movement of DNA fragments in a gel is inversely proportional, in a geometric relationship, to their length. Therefore, the analysis time of longer DNA fragments can be often be untenable. Sample recovery of DNA fragments separated on a gel is difficult and time consuming, requiring specialized techniques.
In addition to the deficiencies of denaturing gel methods mentioned above, these techniques are not always reproducible or accurate since the preparation of a gel slab and running an analysis can be highly variable from one operator to another. As a result, the mobility of a DNA fragment is often different on different gel slabs and even in one lane, compared to another on the same gel slab. The problems and deficiencies of gel based DNA separation methods are well known in the art and are described in “Laboratory Methods for the Detection of Mutations and Polymorphisms”, ed. G. R. Taylor, CRC Press (1997).
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a sensitive and reproducible method which would enable the detection of small amounts of mutant DNA in the presence of a relatively large amount of wild type DNA, wherein such mutations would otherwise go undetected. It is a further object of the invention to provide an analytical method which is reproducible, reliable, inexpensive, can be automated and can be used for high throughput sample screening.
In one aspect, the present invention is a method for detecting a putative mutant DNA in a sample of DNA, the method including the steps of (a) amplifying the sample of DNA using PCR, (b) hybridizing the amplified sample to form a mixture of homoduplexes and heteroduplexes, (c) separating the product of step (b) into fractions by Denaturing Matched Ion Polynucleotide Chromatography, and (d) blind collecting the fractions from step (c) at a retention time corresponding to the retention time of the heteroduplex. The method preferably includes amplifying the fractions collected in step (d) using PCR to obtain an increased amount of heteroduplex relative to homoduplex. The method can also include repeating steps (a) through (d); in a preferred method these steps are repeated until the relative amount of mutant to wild type DNA is increased by an enhancement factor of at least 10 to 1000. The DNA sample can contain a large background of wild type. The putative mutant DNA can be below the limit of detection. The identity of the heteroduplex can be confirmed using standard methods. In a preferred embodiment, the DNA sequence of the wild type DNA and the mutant DNA are known. In a typical analysis using the method of the invention, the mutant DNA differs from wild type DNA by at least one base pair. In a preferred embodiment, the same PCR primers are used to amplify both the mutant DNA and the wild type DNA in the sample. In another preferred embodiment, the retention time used in the blind collection of the heteroduplex in step (d) was previously determined from a reference standard. A preferred reference standard is obtained by separating a standard mixture of homoduplex and heteroduplex, having the same base pair sequence as the sample, by Matched Ion Polynucleotide Chromatography.
In a preferred embodiment of the invention, the separation of the product by Denaturing Matched Ion Polynucleotide Chromatography is effected with an MIPC column containing a stationary phase separation media, and the column is treated before the separating step with a solution for removing any residual DNA from prior separations. For example, the column can be treated before the separating step with from 50 &mgr;L to 1 ml of tetrasodium EDTA adjusted to a pH of 13 with sodium hydroxide. Other treatments for washing a column can also be used alone or in combination with those indicated hereinabove. These treatments include exposing the separation medium to high concentrations of organic solvent (e.g., up to 100% acetonitrile) or exposing the medium to denaturants such as urea or formamide. The column can also be treated by reverse flushing with a washing solution.
In another aspect, the present invention is a method for screening a tissue sample for cancerous cells by detecting a putative mutant DNA in the DNA of the sample, the method including the steps of (a) amplifying the sample DNA using PCR, (b) hybridizing the amplified sample to form a mixture of homoduplexes and heteroduplexes, (c) separating the product of step (b) into fractions by Denaturing Matched Ion Polynucleotide Chromatography, and (d) blind collecting the fractions from step (c) at a retention time corresponding to the retention time of the heteroduplex.
The method preferably

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