Method for isolating nucleic acids
A nucleic acid and chain nucleic acid technology, applied in the field of separation and/or purification of nucleic acids, can solve the problems of concentrated fetal DNA, poor purification of fetal nucleic acid, and inability to capture short-chain nucleic acids well
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Embodiment 1
[0102] Example 1: Test to determine the average length of fetal and maternal DNA
[0103] The size distribution of free circulating DNA was studied using three different plasma pools produced from the blood of women pregnant with boys and babies. The plasma pools are pools A, B, and C. Pool A contains plasma from blood samples of pregnant women from the first trimester to the last trimester of pregnancy. In each case, the pools B and C contain plasma taken from the blood of pregnant women from the first trimester to the second trimester; the content of free circulating fetal DNA is still low at this time, but it is important for clinical diagnosis Higher correlation.
[0104] The starting material used was 10 ml plasma, in the case of pool A 5 ml plasma. The process is the QIAamp blood DNA Midi protocol (QIAGEN) adjusted to a volume of 10 ml. In each case, 300 μl of AE buffer (commercially available from Qagan Company) was used for elution. After elution, ethanol / sodium acetat...
Embodiment 2
[0109] To simulate the different size distributions of fetal DNA (mostly shorter than 300 bp, see Example 1) and maternal DNA (mostly longer than 500 bp), two different PCR amplicons were added to the plasma as a background. The 219bp fragment mimics fetal DNA, and the 1018bp fragment mimics maternal DNA. In the first experiment, a higher content of the PCR amplicon was used for this purpose, that is, 2×10 in each case 6 Copy / 600μl plasma. The process is shown in the following scheme:
[0110] In a 5 ml container, add 90 μl protease (Qiagen Company) and 600 μl buffer AL (Qiagen Company, commercially available) containing guanidine to 600 μl plasma. After vortexing and mixing, the mixture was incubated at 56°C for 15 minutes. After lysis, PCR amplicons with a length of 219 or 1018 bp are added to the lysis buffer. The binding conditions were adjusted with 100 μl of isopropanol, so that the concentration of isopropanol in the total sample was 6.9% (w / v).
[0111] Vortex to mix, t...
Embodiment 3
[0121] The process is as described in Example 2, but this time only 200,000 copies of certain fragments are used to simulate a more realistic scenario of the actual free circulating copy number in the blood. This time, 100 μl of buffer B11 (see above) + 1.2 ml of buffer B6 were used to perform matrix binding under the first condition, thus obtaining an isopropanol concentration of 20.3% by volume (see above). For other conditions of binding to the matrix, 100 μl of buffer B11 and 2.0 ml of buffer B6 were added to the plasma lysate. In addition, under the two above-mentioned buffer conditions, the DNA fragments in the binding mixture in each case were bound to MagAttract matrix or QIAamp mini-column.
[0122] The procedure of the experiment is as follows: add buffers B11 and B6 to the supernatant, mix, and incubate at room temperature for 10 minutes. The two samples were combined and vacuum applied to the QIAamp mini-column (QIAGEN) by means of a telescopic tube (QIAGEN). Wash w...
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