PCR method for overcoming lagging pollution
A lagging, polymerase technology, applied in the field of molecular biology, which can solve the problems of complex operation and inability to clone the product.
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Embodiment 1
[0035] Example 1 Use a pair of primers to amplify a fragment of the GPT-binding protein RhoA gene (gene bank number L25080), and then dilute the PCR amplification product as the amplification template for the next round of PCR reaction. Before the new round of PCR reaction, use one restriction endonuclease or two restriction endonucleases that have a cutting site for the fragment sequence, and use the enzyme that has been completely inactivated by heating as a control. According to the cycle number of the first appearance of the new amplified product band, the degree of damage to the substrate DNA by the restriction endonuclease is estimated. The sequence of forward and reverse primers is shown in Table 3.
[0036] name
[0037]The length of the amplified product is 590bp, and the restriction endonucleases EcoRV and PvuII each have a cutting point for the amplified product fragment. First, commercial human tissue total RNA was used as the original template, and cDNA...
Embodiment 2
[0042] In Example 2, a similar analysis was performed using the human beta-2-adrenoceptor gene (GenBank No. M15169) and human glyceraldehyde-3-phosphate-dehydrogenase gene (GenBank No. XM_006959) as examples. The primer sequence is shown in Table 5.
[0043] name
Location
Sequential (5' to 3')
BAR positive
1574-
1589
CCA GAC TGC GCG CCA T
BAR reverse
1805-
1825
GAT CAG CAC AGG CCA GTG AAG
GPH Positive
553-591
CTG GCC AAG GTC ATC CAT GAC AAC TTT
GGT ATC GTG GAA
GPH reverse
904-940
CGC TGT TGA AGT CAG AGG AGA CCA
CCT GGT GCT CAG T
[0044] The amplified product of the adrenoceptor gene is 252 bases in length, and is simultaneously treated with two kinds of restriction endonucleases PstI and HaeII; The enzymes XhaI and HaeII act. cDNA preparation, first round and second round ...
Embodiment 3
[0047] Example 3 A pair of primers were designed with the human actomyosin gene as the target gene (GenBank No. BC016045). The forward and reverse primers matched the sequence of the target gene, but a mismatched base was introduced respectively, and the forward primer caused an EcoRI cleavage site , while the antiprimer creates a HindIII cleavage site. The sequences and properties of the primers used in Example 3 are shown in Tables 7 and 8.
[0048] name
[0049] The italicized bases in the primers "A forward" and "A reverse" are the introduced mismatched bases, and the underlined parts are the recognition sequences of EcoRI and HindIII formed after the introduction of the mismatched bases. Primer "A1 positive" is the same as the 3' end of primer "A positive"; primer "A1 anti" is the same as the 3' end of primer "A anti".
[0050]
[0051] Oligo(dT) was used to synthesize cDNA, and the reagents and methods for amplifying cDNA with primer "A" were the sam...
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