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Method for improving PCR amplification sensitivity by using integrated host factor

A technology that integrates host factors and sensitivity, applied in the field of nucleic acid molecule amplification technology, can solve the problem that the effect of PCR amplification sensitivity is not very significant, and achieve the effect of improving sensitivity and PCR amplification efficiency

Active Publication Date: 2015-05-06
华桥生物工程科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] U.S. Patent 5,646,019 discloses "a method for preparing nucleic acid templates conducive to priming and amplification", which is to add a heat-resistant single-strand binding protein (SSB, single-stranded nucleic acid binding protein) to the PCR system, SSB The protein only binds to single-stranded DNA, and optimizes PCR amplification by binding and inhibiting the amplification of non-specific fragments containing single strands, but its effect on improving the sensitivity of PCR amplification is not very significant

Method used

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  • Method for improving PCR amplification sensitivity by using integrated host factor
  • Method for improving PCR amplification sensitivity by using integrated host factor
  • Method for improving PCR amplification sensitivity by using integrated host factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: the preparation of IHF

[0023] 1. Insert IHFα or IHFβ subunits derived from Escherichia coli into PET22b expression vectors respectively, and through ligation transformation and colony PCR, enzyme digestion identification and sequencing verification, the recombinant expression vectors PET22-IHFα or pET22-IHFβ can be obtained;

[0024] 2. Transform Escherichia coli BL21 with the constructed recombinant vector pET22-IHFα or pET22-IHFβ, take the positive strain containing the recombinant plasmid and add it to LB liquid medium for overnight cultivation, and then inoculate the bacterial liquid into 50ml of LB culture medium at a ratio of 1% cultured in a shaker at 37°C until the OD value is 0.6-0.8; add IPTG, put the bacterial liquid in a shaker at 37°C to induce expression for 3 hours, and the shaker speed is 160rpm;

[0025] 3. Centrifuge the bacterial solution after the induced expression to obtain the bacterial cells, lyse and release the protein. Protein...

Embodiment 2

[0046] Embodiment 2: Optimization of PCR reaction

[0047] Use pUC19-IHF as template for PCR amplification.

[0048] Primer 1 (as shown in SEQ ID NO.2 in the sequence listing):

[0049] 5'-TATGACCAAGTCAGAATTGATA-3';

[0050] Primer 2 (as shown in SEQ ID NO.3 in the sequence listing):

[0051] 5'-CCGTAAATATTGGCGCGATCGCGC-3'.

[0052] Prepare 20 μl of PCR reaction solution according to the following table:

[0053] Reagent

Usage amount (μl)

Final concentration

2×Phusion Master Mix

10

1×

Primer1

0.1

0.018μM

Primer2

0.1

0.017μM

template DNA

0.2

0.78ng

Sterile distilled water

9.6

total capacity

20

[0054] Carry out PCR reaction according to the following conditions: 95°C for 60s. 95°C for 30s, 55°C for 30s, 72°C for 30s, a total of 30 cycles. Finally, 72°C for 5 minutes.

[0055] In the PCR reaction solution, different concentrations of the IHF protein obt...

Embodiment 3

[0058] Embodiment 3: Optimization of nested PCR reaction

[0059] ①Take PFE-2-MA as a template for the first PCR

[0060] Primer 1 (shown as SEQ ID NO.4 in the sequence listing):

[0061] 5'-CTGCCGTTCATCGTTGGTGGTCGTGACGTTTCTCC-3';

[0062] Primer (shown as SEQ ID NO.5 in the sequence listing) 2:

[0063] 5'-AGAGGTAACGCAACCAGAACGACCCCAAAGAG-3'.

[0064] Prepare 20 μl of PCR reaction solution according to the following table:

[0065]

[0066] The PCR reaction was carried out according to the following conditions: 95°C for 30s, 58°C for 30s, 72°C for 50s, a total of 30 cycles. Finally, 72°C for 5 minutes.

[0067] ② Use the first PCR product diluted 100 times as the template for PCR amplification, and perform the second PCR

[0068] Primer 1 (as shown in SEQ ID NO.6 in the sequence listing):

[0069] 5'-CCTGCGCTGCTCACTGCTCTTCTGACTCTC-3';

[0070] Primer 2 (as shown in SEQ ID NO.7 in the sequence listing):

[0071] 5'-GTGGTGCATTCAGCTTCGGTCAGGATGTCA-3'.

[0072] Prepa...

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Abstract

The invention provides a method for improving PCR amplification sensitivity by using an integrated host factor. The method comprises the following steps: adding the integrated host factor IHF into a reaction solution containing deoxyribonucleoside triphosphate, DNA polymerase, an oligonucleotide primer and a nucleic acid fragment which serves as a template, carrying out repeated heat circulation according to three steps of unwinding, annealing and extending of the normal PCR, thereby amplifying a target nucleic acid fragment, wherein the mole ratio of a primer to the integrated host factor IHF is 0.005-1. The method is capable of specifically and efficiently amplifying a target nucleic acid sequence.

Description

【Technical field】 [0001] The invention relates to a method for nucleic acid molecule amplification technology based on polymerase chain reaction (PCR), including linear unit, multiplex and nested PCR reactions, and mainly relates to a method using Thermostable, integrated host factor (IHF) that binds to DNA double strands to improve the efficiency of PCR amplification. 【Background technique】 [0002] PCR is a molecular biology technique used to amplify a specific segment of DNA, which can be performed outside the body and does not have to rely on organisms such as E. coli or yeast. Polymerase chain reaction technology is widely used in medical and biological laboratories, for example, it is used to determine whether a certain genetic disease will appear in the sample, the diagnosis of infectious diseases, gene replication, and paternity testing, etc. [0003] In practical applications, especially when using the existing PCR technology to diagnose infectious diseases, there ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2522/101C12Q2531/113C12Q2549/00
Inventor 戚智青唐黎张秀英侯丹刁勇齐晓雪许瑞安马国兴杜民
Owner 华桥生物工程科技有限公司
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