PCR method of universal primer and its reaction liquid and application in multiple PCR

A universal, chain reaction technology, applied in the field of molecular biology, can solve problems such as large differences, inconsistent PCR amplification efficiency, and inability to produce products, so as to improve accuracy, avoid false negatives or incorrect semi-quantitative judgments Effect

Inactive Publication Date: 2004-07-21
徐定邦 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Existing multiplex PCR uses multiple pairs of primers that match the base sequence of the target template to simultaneously add to a PCR reaction tube to achieve multiple amplification. During the entire PCR amplification process, primers that are complementary to the original template are always used. In fact, , each pair of primers is paired with the template and then extended, each PCR is not related to e

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Design of universal primers: design the following primers (Table 1) according to the principles of universal primer design in the technical scheme.

[0020] Universal Primer U1

Embodiment 2

[0022] Universal primer-based polymerase chain reaction.

[0023] Leading primers were designed against human actomyosin and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene sequences (Table 2).

[0024] 8A1U1(5')

CCC CCC CCC CGC CCC CCC GCT ACG TCG CCC TGG ACT TC

8A2U1(3′)

CCC CCC CCC CGC CCC CCC CCG CCA GAC AGC ACT GTG TT

8G1U1(5′)

CCC CCC CCC CGC CCC CCC CGA CAG TCA GCC GCA TCT TC

8G2U1(3')

CCC CCC CCC CGC CCC CCC ACG TAC TCA GCG CCA GCA TC

8G3U2(5′)

CCC CCC CCC CCC TCC CCC CCC TTT TAT GGC ACC GTC AAG

GCT GA

8G4U2(3')

CCC CCC CCC CCC TCC CCC CCC TTT TAG TGA TGG CAT GGA

CTG TGG TC

[0025] Reaction sequence

No

F1

F2

F3

F4

F5

F6

F7

F8

F9

F10

8A1U1

8A2U1

0.005

0.01

...

Embodiment 3

[0029] Application in multiplex PCR.

[0030] Two pairs of leading primers 8A1U1(5') / 8A2U1(3') and 8G1U1(5') / 8G2U1(5') were simultaneously added to a PCR reaction tube at a concentration of 0.01 uM. The universal primer 8U1 was added at a concentration of 5uM, and the other reaction conditions were the same as in Example 2. As a result, two amplified bands were obtained, which were 292 and 377 bp respectively.

[0031] Nucleotide sequence

[0032] Human actomyosin gene sequence

[0033] 1 accgcgtccg ccccgcgagc acagagcctc gcctttgccg atccgccgcc cgtccacacc

[0034] 61 cgccgccagc tcaccatgga tgatgatatc gccgcgctcg tcgtcgacaa cggctccggc

[0035] 121 atgtgcaagg ccggcttcgc gggcgacgat gccccccggg ccgtcttccc ctccatcgtg

[0036] 181 gggcgcccca ggcaccaggg cgtgatggtg ggcatgggtc agaaggattc ctatgtgggc

[0037]241 gacgaggccc agagcaagag aggcatcctc accctgaagt acccccatcga gcacggcatc

[0038] 301 gtcaccaact gggacgacat ggagaaaatc tggcaccaca ccttctacaa tgagctgcgt

[0039] 361 gtgcctcc...

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PUM

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Abstract

A PCR method for universal primer, its reaction liquid, and its application in multiple PCR are disclosed. Said PCR method features that a pair of leader primers complementary with template in region 3' and a pair of universal primers the same as said leader primer in region 5' are amplified in a single PCR. Its advantages are high reaction specificity, stable amplifying efficiency and wide concentration range of primer.

Description

technical field [0001] The invention relates to molecular biology technology, in particular to a polymerase chain reaction method, its reaction liquid and its application in multiple PCR. technical background [0002] PCR is a simple, specific, sensitive, and rapid gene amplification method, and its process usually includes 20-40 cycles consisting of three steps of denaturation, annealing, and extension. The key to the PCR method is to design a pair of primers with high stringency that are complementary to the two strands of the DNA of the target gene according to the sequence of the target gene. The double-stranded DNA is unwound into two single strands at the denaturing temperature. When the temperature drops to the annealing temperature, the forward and reverse primers are respectively combined with the two strands of the template DNA, and then at the extension temperature, the DNA polymerase starts at the 3' end where the primer and the template bind. One cycle of ampli...

Claims

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Application Information

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IPC IPC(8): C12P19/34C12Q1/68
Inventor 徐定邦朱德芬陈有容徐文慧
Owner 徐定邦
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