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Fetal free DNA (Deoxyribonucleic Acid) library quantitative standard and preparation method therefor

A DNA library and standard product technology, applied in the field of biological products, can solve the problems of increasing costs and affecting the efficiency of prenatal screening and diagnosis of chromosomal diseases, etc., and achieves easy preservation and small variation coefficient of qPCR standard product preparation bottles , to avoid the effect of bias

Active Publication Date: 2016-01-13
GUANGZHOU DARUI BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are almost no qPCR quantitative standards for library DNA concentration involved in this detection method on the market, which seriously affects the efficiency of prenatal screening and diagnosis of chromosomal diseases and increases the cost

Method used

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  • Fetal free DNA (Deoxyribonucleic Acid) library quantitative standard and preparation method therefor
  • Fetal free DNA (Deoxyribonucleic Acid) library quantitative standard and preparation method therefor
  • Fetal free DNA (Deoxyribonucleic Acid) library quantitative standard and preparation method therefor

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: the preparation of standard substance

[0037] 1. Adapter sequence and primer design and synthesis

[0038]A sequence that was as inconsistent as possible with the human genome was designed, and the following linker sequences were obtained according to the GC content and primer design principles. In order to prevent the linker sequence from being connected to the target fragment in the reverse direction, a sticky end is set at the 3' end, and the protruding C is modified to prevent the sticky end from being degraded. Both ends of the 5' were dephosphorylated. In order to prevent the blunt end of the adapter sequence itself from ligation, the following factors were combined to synthesize the following double-stranded adapter sequences AdapterS1 and AdapterS2:

[0039] Adapter S1:

[0040] 5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3′

[0041] 3′-C*C*C*GGTAGAGTAGGGACGCACAGAGGCTGAGTC-5′

[0042] Adapter S2:

[0043] 5′-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT-3′...

Embodiment 2

[0063] Embodiment 2: the use method of standard substance and the establishment of standard curve

[0064] 1. Dilution method

[0065] Dilute the obtained library according to the following calculation method, the first dilution to 100pM

[0066]

[0067] 660 (Da) is the relative molecular mass of one base pair of DNA.

[0068] The 100pM library was sequentially diluted 10-fold to obtain 10pM, 1pM, 0.1pM, 0.01pM and 0.001pM libraries.

[0069] 2. Experimental method

[0070] The 6 library dilutions obtained in step 1 were subjected to real-time fluorescent quantitative PCR. The KAPASYBRFAST Universal qPCR kit was used for analysis.

[0071] qPCR reaction system: 20 μL mixture contains 4 μL of template, 10 μL of 2×SYBR mix, 1 μL (10 μM) of primers NIPT-LibraryF and primer NIPT-LibraryR, and 4 μL of NucleaseFreeWater.

[0072] qPCR reaction program: pre-denaturation at 95°C for 5 min, 40 cycles of denaturation at 95°C for 30 s, and annealing at 60°C for a total of 45 s. ...

Embodiment 3

[0078] Example 3: Repeatability of qPCR Quantitative Standard Detection

[0079] The prepared 100pM standard and the other 6 dilutions of the standard were repeatedly frozen and thawed 8 times at the same time for qPCR detection. The CT value of the number of cycles obtained is shown in Table 2. The coefficient is less than 5.00%, relatively small, and can be used in conjunction with a trisomy 21, 18, 13 trisomy detection kit (semiconductor sequencing method) (8 RUNs).

[0080] Table 2. Results of 8 repeated freeze-thaw cycles

[0081]

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Abstract

The invention discloses a qPCR (quantitative Polymerase Chain Reaction) quantitative standard for detecting library DNA (Deoxyribonucleic Acid) concentration of aneuploids, i.e., 21 trisome, 18 trisome and 13 trisome of fetal chromosomes based on a semiconductor sequencing method and a preparation method therefor and belongs to the field of noninvasive prenatal detection on fetal aneuploids. The standard is used for carrying out quality control on detection concentration of detected samples. The preparation method for the qPCR quantitative standard disclosed by the invention comprises five steps: (1) synthesizing joint sequences and primers; (2) preparing simulated fetal free DNA libraries; (3) amplifying and purifying the libraries; (4) producing a library standard curve; and (5) carrying out sample detection. According to the method disclosed by the invention, the DNA concentration of sample libraries can be rapidly determined, and a guarantee of accuracy and validity of noninvasive prenatal detection on the aneuploids, i.e., the 21 trisome, the 18 trisome and the 13 trisome of the fetal chromosomes is provided.

Description

technical field [0001] The invention belongs to the technical field of biological products, belongs to the category of non-invasive prenatal detection of fetal chromosomal aneuploidy, and relates to qPCR for detecting the library DNA concentration of fetal chromosomal aneuploidy trisomy 21, trisomy 18 and trisomy 13 by semiconductor sequencing method Quantitative standards and methods for their preparation. Background technique [0002] Chromosomal aneuploidy refers to the increase or decrease in the number of one or several chromosomes in a cell compared to the normal 46 chromosomes in humans, which is closely related to significant morbidity and mortality in infants and young children. The incidence of such diseases increases with the increase of maternal age. According to literature reports, about 100,000 newborns with chromosomal abnormalities are born in my country every year, and chromosomal abnormalities account for 0.3% of live babies. Among them, trisomy 21, trisom...

Claims

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Application Information

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IPC IPC(8): C40B50/06C40B40/06C12N15/10
Inventor 李明高旭年杨学习李尔华杨旭雷孝锋
Owner GUANGZHOU DARUI BIOTECH
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