Method for assaying DNA fragments in mixture

Details Method for assaying DNA fragments in mixture Method for assaying DNA fragments in mixture

1999-10-07

2001-05-01

Jones, W. Gary (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091200, C435S173300, C435S320100, C536S023100, C536S025400

Reexamination Certificate

active

06225064

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to a method for amplifying nucleic acid, a method for assaying DNA fragments in mixture, a method for assaying RNA fragments in mixture, and an expression profiling process of a group of expressed genes; more specifically, the invention relates to a method for assaying DNA fragments by gel electrophoresis and by using fluorophore label.
The nucleotide sequence of a gene or a DNA can be compared with other nucleotide sequences by DNA sequencing, but it is very difficult to determine the sequence of a long DNA or a mixture sample of a great number of DNA fragments. So as to determine the sequence of such long DNA, therefore, the long DNA is first fragmented; then, the resulting DNA fragments are assayed on gel electrophoresis patterns. Due to the recent progress of various assay methods and apparatuses therefor, numerous expressed genes can simultaneously be detected and assayed.
The expression profiling method includes for example a scanning process based on gel electrophoresis. The scanning process includes FDD [Fluorescent Differential Display; FEBS Letters 351, 231-236 (1994)] and a process using terminal nucleotide selective primer [Nucleic Acids Research, 24, 2616-2617 (1996); Nucleic Acids Symposium Series, No. 35, 257-258 (1996)].
All these scanning processes comprise PCR (Polymerase Chain Reaction) using plural primers in common to plural DNA fragments along with a sample cDNA or mRNA as template and recovering gene expression information based on the electrophoresis patterns of the resulting PCR products. Because the expression can be detected with no use of any gene-specific probe according to the scanning processes, advantageously, the expression profile of a gene with a nucleotide sequence not yet identified can be yielded.
The process using terminal nucleotide selective primer comprises digesting a double-stranded cDNA with a restriction enzyme and ligating an oligonucleotide with a known nucleotide sequence at the terminus of a DNA fragment as a digestion product. PolyA tail is generally present at the 3′ terminus of cDNA. Because a nucleotide sequence between the polyA tail at the 3′ terminus of the cDNA and the ligated oligonucleotide is specific to the cDNA, a DNA fragment carrying the nucleotide sequence is assayed as a fragment representing (or identifying) the cDNA.
Because gene species of several thousands to several ten thousands in number are expressed in living organisms, the types of such cDNA representative fragments are so numerous that these representative fragments cannot be separated and assayed in one lane by electrophoresis. Thus, all these types of such representative fragments are divided in plural groups, whereby the number of such DNA fragments included in each one group is sufficiently reduced such that the DNA fragments in each one group can be separated and assayed satisfactorily by gel electrophoresis. So as to divide all the representative fragments in plural groups, PCR primers with a selective nucleotide sequence composed of two nucleotides at the 3′ terminus thereof are used. By PCR, DNA fragments with terminal two nucleotides complementary to each selective nucleotide sequence are amplified. From two primer sets with two selective nucleotide sequences is selected each one primer; subsequently, a group of a combination of appropriate two primers is prepared.
By PCR with primers of the individual sets, the resulting amplified products are assayed by electrophoresis. Via combinations of DNA fragments expressed on the electrophoresis pattern recovered by using the primers of the individual sets, the whole information of each expressed gene can be recovered. Consequently, the type of each expressed gene and the expression level thereof can be identified.
For fragment assay, at least one primer of oligo dT primer and a primer complementary to the oligomer ligated at the terminal digestion site of a DNA fragment is labeled with fluorophore; using the primer labeled with the fluorophore, the DNA fragment is amplified in such a number above the detection sensitivity of an assay apparatus such as fluorescent DNA sequencer and the like.
SUMMARY OF THE INVENTION
Conventional expression profiling processes based on electrophoresis have various drawbacks as described below in practical sense. By the processes on the basis of electrophoresis, the resulting comparative results are at serious error if the reproducibility of PCR amplification is low.
For comparing the difference in expression profile between plural samples, the samples each are independently subjected to PCR and the resulting PCR products are then electrophoresed. The results are compared to each other. Plural samples labeled with the same fluorophore species are subjected to PCR in different reaction tubes; and the resulting PCR products are assayed in different electrophoresis lanes. Plural samples labeled with different fluorophore species are independently subjected to PCR in different tubes; and the resulting products are assayed in one electrophoresis lane. According to the conventional processes, sample preparation for electrophoresis requires PCR in different tubes, so PCR reproducibility influences the precision of the comparison of the electrophoretic assay results of PCR products. Currently, nevertheless, PCR reproducibility is disadvantageously insufficient.
It is an object of the present invention to provide a method for assaying DNA fragments in mixture, comprising PCR in one reaction tube, thereby enabling an expression profiling process of comparing plural samples together, under no influence of PCR reproducibility.
According to the inventive method for assaying DNA fragments in mixture, each sample requires a PCR primer with a different nucleotide sequence but of the same length and the same melting temperature (T
m
), for reaction of plural samples in one tube. Each primer corresponding to each of plural samples is of a nucleotide sequence, with no chance of secondary structure formation between these primers, so that these primers can independently function and never influence the reaction of other primers. Plural DNA samples are placed in one reaction tube for PCR, whereby variation of each PCR can be eliminated.
So as to amplify a first DNA sample with a primer of a first primer set and amplify a second DNA sample with a primer of a second primer set, an oligonucleotide with a complementary nucleotide sequence to each primer is ligated to the primer. So as to identify the primers serving for amplification, the primer of the first primer set and the primer of the second primer set are labeled with different fluorophore species. Because plural DNA samples are amplified by using the primers of plural primer sets in one tube, no problem concerning PCR reproducibility occurs.
In order to compare together DNA fragments amplified with the primers of plural primer sets, the difference in reaction reactivity between these primers should be eliminated. T
m
value of primer, T
m
value of sample DNA, and T
m
value of PCR amplified product determine the PCR reaction efficiency.
Expression of one gene in plural sample DNAs means that the sample DNAs with relation to the gene and the PCR amplified products thereof are identical; thus, the T
m
values thereof are equal. Hence, the PCR reaction efficiency depends on the T
m
value of primer. By setting the T
m
values of plural primers at an equal value, the resulting PCR reaction efficiencies can be retained equally.
The T
m
value of DNA can be calculated, approximately, depending on the nucleotide species composing the DNA sequence [Biopolymers, 3, 195-208 (1965)]. The T
m
value can be calculated by using the difference in stacking between a nucleotide species and a nucleotide adjacent thereto. By using a nucleotide sequence composed of an interesting nucleotide and one nucleotide adjacent thereto, the T
m
value can be more accurately calculated [Proc. Natl. Acad. Sci. USA, 83, 3746-3750 (1986)].
So as to pre

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