Method for performing multi-target-site amplification library construction on plasma free DNA (deoxyribonucleic acid)

A multi-target site and plasma technology, applied in the field of multi-site amplification library for plasma free DNA or short fragment DNA, to avoid primer complementation and mismatch, reduce initial requirements, and simplify primer design

Active Publication Date: 2016-03-30
HANGZHOU GUKUN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The technical problem to be solved by the present invention is to provide a method for multi-target site amplification and library construction of short fragment DNA such as plasma free DNA. In view of the defects of existing mul

Method used

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  • Method for performing multi-target-site amplification library construction on plasma free DNA (deoxyribonucleic acid)
  • Method for performing multi-target-site amplification library construction on plasma free DNA (deoxyribonucleic acid)
  • Method for performing multi-target-site amplification library construction on plasma free DNA (deoxyribonucleic acid)

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Experimental program
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Embodiment 1

[0048] Embodiment 1, carry out multi-target site amplification method for building a library of plasma cell-free DNA, the following steps are carried out in sequence:

[0049] 1), end repair and add A

[0050] Prepare the reaction mixture according to the ratio in Table 1 below, and use a gun to gently blow up and down to mix evenly. Ultra TM IIEndRepair / dA-TailingModule (E7546) includes two parts: NEBNextUltraIIEndPrepEnzymeMix and NEBNextUltraIIEndPrepReactionBuffer, and the initial amount of free DNA is in the range of 500pg-lug (for example, 10ng).

[0051] Table 1

[0052] NEBNext Ultra II End Prep Enzyme Mix

3ul

NEBNext Ultra II End Prep Reaction Buffer

7ul

Cell-free DNA obtained from the above preparatory steps

50ul

Total

60ul

[0053] Place it in the PCR instrument and set the program reaction:

[0054] Set the top cover temperature to ≥75°C

[0055] 20℃30min

[0056] 65℃30min

[0057] Store at 4°C;

[0058] A ...

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Abstract

The invention discloses a method for performing multi-target-site amplification library construction on plasma free DNA (deoxyribonucleic acid). The method sequentially comprises steps as follows: 1), tail end repairing and A addition are performed; 2), linker connection is performed: a linker DNA with an annular hairpin structure formed through annealing is added to a DNA fragment with A addition at the tail end in the step 1), so that double-stranded DNA after being combined with annular hairpin linkers on two sides is obtained; when the amount of plasma free DNA extracted and purified in the step 1) is smaller than or equal to 10 ng, a step 3) is performed; otherwise, when the amount of plasma free DNA extracted and purified in the step 1) is larger than 10 ng, a step 4) is performed; step 3), a specific primer I, Phi29 enzyme and a buffer solution are added to DNA with linker addition in the step 2), and linear amplification is performed; step 4), PCR (polymerase chain reaction) amplification is performed: amplification products constitute a free DNA mutation site enrichment sequencing library.

Description

technical field [0001] The invention belongs to the fields of molecular biology and medicine, and in particular relates to a method for multi-site amplification library of plasma free DNA or short fragment (<300bp) DNA. Background technique [0002] In recent years, it has been found that there are free DNA fragments in the peripheral blood plasma of the human body, with a length of 100-300bp, mainly concentrated around 150bp. In cancer patients, these cell-free DNA may include shed and dead tumor cells, and in pregnant women, may include fetal DNA fragments. Therefore, the mutation of a specific site in a tumor or fetus can be reflected to a certain extent by detecting the mutation of a specific site in the cell-free DNA fragment. However, the free DNA fragments in plasma are relatively short, most of them are around 150-200bp, generally not more than 300bp, and the content is very low. The extraction rate of existing free plasma DNA extraction methods is generally 10n...

Claims

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Application Information

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IPC IPC(8): C40B50/06
CPCC40B50/06
Inventor 金谷雷牛耀芳
Owner HANGZHOU GUKUN BIOTECH CO LTD
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