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A dna molecular weight standard fragment amplification single-stranded primer, amplification method and preparation method of dna molecular weight standard

A molecular weight standard, single-stranded technology, used in the preparation of DNA molecular weight standards, the field of DNA molecular weight standard amplification fragment single-stranded primers, can solve the problems of unfavorable purification, large-scale production, residual template, high cost, and avoid non-specificity. The effect of banding, improving efficiency, and reducing amplification cost

Active Publication Date: 2022-04-19
北京擎科生物科技股份有限公司
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Problems solved by technology

[0005] However, for small fragments in DNA markers, especially 100bp DNA, due to the small fragment length, the cost of PCR amplification is high, and the method of using restriction plasmids often amplifies non-specific bands, And the template has residues, etc., which is not conducive to subsequent purification and large-scale production

Method used

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  • A dna molecular weight standard fragment amplification single-stranded primer, amplification method and preparation method of dna molecular weight standard
  • A dna molecular weight standard fragment amplification single-stranded primer, amplification method and preparation method of dna molecular weight standard
  • A dna molecular weight standard fragment amplification single-stranded primer, amplification method and preparation method of dna molecular weight standard

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preparation example Construction

[0041] The preparation method of the DNA molecular weight standard provided by the present invention, the DNA molecular weight standard includes a DNA fragment with a length of 100bp, and the DNA fragment is obtained by the above-mentioned amplification method; after the DNA fragment of 100bp is obtained, it is mixed with DNA fragments of other lengths, Get the DNA marker.

[0042] Preferably, the DL2000 DNA marker can be prepared by the preparation method of the present invention.

Embodiment

[0044] In this embodiment, a single-stranded primer with a length of 60 bp is used for amplification, and its 3' end has a palindromic sequence of 20 bp.

[0045] The single-stranded primer sequence in this embodiment is shown in SEQ ID No: 1, specifically:

[0046] 5`-TCCGATCGAATTCAATTAGAATTCAGCTAGAATTCATTCAGAATTCGCGGCCGCGAATTC-3`;

[0047]Wherein, the palindrome sequence is GAATTCGCGGCCGCGAATTC(-3'), and the GC content is 50%.

[0048] Using the above single-stranded primers for PCR amplification, the reaction system is:

[0049] 45 μl gold medal PCR mix, 5 μl primers; reaction program: 98°C for 90s, 98°C for 10s, 58°C for 10s, 72°C for 10s, 72°C for 1min, 4°C hold, 35 cycles.

[0050] In the above cycle process, the specific replication process of the primer is:

[0051] The first round of amplification is carried out first, and the specific process is as follows: figure 1 As shown, one single-stranded primer molecule and another single-stranded primer molecule are anne...

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Abstract

The invention relates to a DNA molecular weight standard fragment amplifying single-strand primer, an amplification method and a preparation method of a DNA molecular weight standard. The 3' end of the single-stranded primer is provided with a palindromic sequence, which enables it to be used as both an upstream and downstream primer as well as a template, thereby avoiding the problem of using separate templates and primers for amplification in the prior art. The problem of template residue is easy to occur in the process, which is beneficial to the efficient purification after amplification; the single-stranded primer of the present invention is suitable for the amplification of small fragments of 50-100bp in DNA markers, which not only reduces the amplification cost, but also improves the efficiency; The preparation method of the invention can improve the large-scale production efficiency of the DNA marker and effectively reduce the cost.

Description

technical field [0001] The invention relates to the field of gene amplification, in particular to a DNA molecular weight standard amplification fragment single-stranded primer, an amplification method and a preparation method of the DNA molecular weight standard. Background technique [0002] DNA molecular weight standard, also known as DNA marker, is a mixed reagent of a series of DNA fragments with different molecular weights. In the field of molecular science, a DNA marker is a reagent used to calibrate the molecular weight of the DNA fragment to be tested; the specific calibration method is to electrophoresis the DNA marker and the DNA fragment to be tested on the same agarose gel, and the different molecular weights in the DNA marker The length fragments form a gradient distribution strip (ladder), and the strip of the DNA fragment to be tested is compared with the DNA ladder to obtain its corresponding length. Among commonly used DNA markers, the minimum DNA length is...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6806C12N15/11
CPCC12Q1/6806C12Q2531/113C12Q2545/114C12Q2565/125
Inventor 马石金汪俭肖晓文谢天崔银芳廖倩杜军
Owner 北京擎科生物科技股份有限公司
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