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A method for selectively amplifying cell-free fetal DNA in maternal plasma

A selective, pregnant woman's technology, applied in the field of DNA amplification, can solve the problems of low DNA content and low accuracy of enriching free fetal DNA molecules, and achieve the effect of improving sensitivity and specificity

Inactive Publication Date: 2016-09-07
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the DNA content in plasma is very small, and the method for enriching free fetal DNA molecules has low accuracy, and it is very easy to bring in exogenous nucleic acid pollutants, the concentration of which is at the ng / ml level, and the free fetal DNA is only the total free DNA in plasma Even if the extraction efficiency can reach 100%, it still takes a few milliliters or even tens of milliliters of blood samples to meet the requirements of routine testing

Method used

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  • A method for selectively amplifying cell-free fetal DNA in maternal plasma
  • A method for selectively amplifying cell-free fetal DNA in maternal plasma
  • A method for selectively amplifying cell-free fetal DNA in maternal plasma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Preparation of PCR amplification reaction template

[0046] (1) Design and synthesis of two oligonucleotides A and B: oligonucleotide A: 5'-GCG GTG ACC CGG GAG ATCTGA ATT CT-3', oligonucleotide B: 5'-GAA TTC AGA TC-3' .

[0047] (2) Oligonucleotide B 5'end phosphate modification, the reaction is as follows (50μl total system):

[0048]

[0049] Mix gently by pipetting, and inactivate in a 37℃ water bath for 60 minutes and 75℃ water bath for 10 minutes.

[0050] (3) Annealing reaction (100μl total system):

[0051]

[0052] The reaction conditions are as follows:

[0053] 95℃ 2min

[0054] 95°C (decrease by 1°C every 90sec to 25°C)

[0055] Keep warm at 4℃

[0056] The resulting product is the linker DNA.

[0057] (4) End-filling of free DNA: Using 100ng of free DNA extracted by the phenol / chloroform / isoamyl alcohol method, perform the following reaction (10μl total system):

[0058]

[0059]

[0060] Purify DNA using AxyPrep PCR cleaning kit and dissolve it in 25μl Eluent s...

Embodiment 2

[0068] Example 2: Selective PCR amplification of fetal free DNA in maternal plasma

[0069] Use the ligation product of Example 1 as a template and oligonucleotide A as a primer for PCR amplification reaction (total system 20 μl):

[0070]

[0071] The PCR amplification reaction process is as follows:

[0072] (a) The joint is extended for 5 minutes at 72℃,

[0073] (b) Pre-denaturation at 80℃ for 3min,

[0074] (c) Denaturation at 80℃ for 20s,

[0075] (d) Annealing at 72°C (each cycle is 0.5°C lower than the previous cycle) for 20s,

[0076] (e) Extension at 72°C for 2min,

[0077] (f) Repeat (c)-(e) 30 cycles,

[0078] (g) Denaturation at 80℃ for 20s,

[0079] (h) Annealing at 57℃ for 20s,

[0080] (i) Extend for 2min at 72℃,

[0081] (j) Repeat (g)-(i) 10-20 cycles,

[0082] (k) Extend at 72℃ for 5min,

[0083] (l) Keep warm at 4°C.

Embodiment 3

[0084] Example 3: Selective PCR amplification of fetal free DNA in maternal plasma

[0085] Use the ligation product of Example 1 as a template and oligonucleotide A as a primer for PCR amplification reaction (total system 20 μl):

[0086]

[0087]

[0088] The PCR amplification reaction process is as follows:

[0089] (a) The 72°C joint is extended for 5 minutes,

[0090] (b) Pre-denaturation at 81℃ for 3min,

[0091] (c) Denaturation at 81℃ for 20s,

[0092] (d) Annealing at 72°C (each cycle is 0.5°C lower than the previous cycle) for 20s,

[0093] (e) Extend for 2min at 72℃,

[0094] (f) Repeat (c)-(e) 30 cycles,

[0095] (g) Denaturation at 81℃ for 20s,

[0096] (h) Annealing at 57℃ for 20s,

[0097] (i) Extend for 2min at 72℃,

[0098] (j) Repeat (g)-(i) 10-20 cycles,

[0099] (k) Extend at 72℃ for 5min,

[0100] (l) Keep warm at 4°C.

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Abstract

The invention relates to a method of selectively amplifying free fetal DNA (deoxyribonucleic acid) in maternal plasma. The method comprises the following steps: performing A adding reaction after carrying out end-filling on the free DNA in the maternal plasma to obtain the free DNA with a sticky end; then connecting the free DNA with linker DNA prepared by utilizing oligonucleotides to react; by taking a connecting product as a template and taking the oligonucleotides as primers to carry out PCR (polymerase chain reaction) amplification reaction, namely extending the linker at 72 DEG C, pre-degenerating for 3 minutes at 80-87 DEG C, degenerating for 20 seconds at 80-87 DEG C, annealing for 20 seconds at 72 DEG C, extending at 72 DEG C, degenerating for 20 seconds at 80-87 DEG C, annealing for 20s at 57 DEG C, extending at 72 DEG C, and preserving the heat at 4 DEG C. The method can be used for selectively amplifying the free fetal DNA without amplifying the maternal DNA, so that the maternal DNA background interference can be eliminated to the greatest extent, and thus, the sensitivity and the specificity of the diagnosis are improved.

Description

Technical field [0001] The invention relates to the technical field of DNA amplification, and specifically refers to a method for selectively amplifying free fetal DNA in pregnant women's plasma. Background technique [0002] There is a small amount of free fetal DNA (Cell-free fetal DNA, cffDNA) in the plasma of pregnant women, which is an ideal test material for non-invasive prenatal diagnosis. The use of free fetal DNA can be used for early and specific prenatal screening and diagnosis, and It is not easy to be interfered by previous pregnancy, which provides a new way for non-invasive prenatal diagnosis. However, the content of free fetal DNA in pregnant women’s plasma is very small, and free fetal DNA is mixed with maternal DNA. Existing studies on free fetal DNA in pregnant women’s plasma are conducted under the interference of high maternal DNA background. Easy to cause errors. [0003] Most free fetal DNA molecular fragments in plasma are less than 300bp in length, and mo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6855C12Q2531/113C12Q2525/191
Inventor 张桂珍杨麒巍杜珍武宋旸于杉
Owner JILIN UNIV