polynucleotide amplification
A technology for polynucleotides and nucleotides, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, etc., and can solve problems such as misreading, restriction sequence search, etc.
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Embodiment 1
[0120] Example 1: Principle of the method
[0121] exist figure 1 An overview of examples of polynucleotide amplification methods according to the present invention is given in . Anchor primers for reverse transcription can conform to the formula dT x V or dT x VN y (defined with V or VN y Repeated x of combined deoxythymidine (“dT”) assigns sufficiently high specificity and melting temperature to successfully initiate RT reactions; V is deoxyguanylic acid (“dG”), deoxyadenosine (“dA”) or deoxycytidine ("dC"); N is a sequence of y nucleotides independently selected from deoxyadenosine, deoxycytidine, deoxyguanosine, or deoxythymidine; if more than one base is used for the anchor primer , y determines their number; increasing y will increase the subpopulation of reverse transcripts. Tailing of the 3' end of such synthesized cDNA with a poly-N tail is enabled by the use of terminal transferase - N represents a homogeneous nucleotide having a poly-N tail selected from deox...
Embodiment 2
[0122] Example 2: Amplification of all RNA molecules present in a sample
[0123] Because all the mRNA molecules present in a sample, even in well-characterized tissues such as those derived from human or mouse liver, are unknown, and their number may be in the tens of thousands, a method was chosen to use a method consisting of 14 A population of synthetic mRNA molecules was used to show that all RNA molecules present in the sample were detected. For this purpose, sequences of genomic DNA of different lengths derived from the mouse GAPDH gene (GenBank accession number: NC000072) were amplified using standard PCR. A 5' primer with an anchored T7 polymerase promoter was used. A combination of 4 bases defining each mRNA at its 5' end is introduced after the promoter. Primers selected for the 3' end included a combination of 4 bases following the GAPDH specific sequence that would define these molecules upstream of the polyA tail. After these four bases, 21 deoxythymidines, or...
Embodiment 3
[0135] Example 3: Amplification of total RNA from mouse liver.
[0136] Total RNA (13 μg) from mouse liver was reverse transcribed, tailed and PCR amplified under the same conditions as in Example 2 for the complete artificial RNA series.
[0137] The same conditions were used for RT, tailing and PCR to show that it is possible to display RNA molecules from complex mixtures under the same stringent conditions that enable the reaction of artificial RNA. The PCR products were run on a 0.7% agarose gel to resolve longer molecules. If you can image 3 As seen in , each primer combination amplified a unique profile.
[0138] exist image 3 The length distribution of the RNA molecules shown in is in good agreement with the length distribution of cDNAs in the NCBI nucleotide database for screening non-redundant full-length mouse cDNA clones ( Figure 5 ).
[0139] Because an average of about 10-15 bands can be seen on each lane, the full set of 768 primer combinations (3×4 4 ) ...
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