Method for screening nucleic acid fragments in predetermined range in nucleic acid sequencing library
A nucleic acid sequencing technology with a predetermined range, applied in the field of biomedicine, can solve the problem of high cost of whole genome sequencing, and achieve the effect of universality, increased concentration and wide application
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Embodiment 1
[0078] Obtain free nucleic acid from a predetermined source in the biological sample to be tested;
[0079] performing end repair on the free nucleic acid to obtain end repaired free nucleic acid;
[0080] Add "A" to the end of the free nucleic acid after terminal repair to obtain the free nucleic acid with "A" added to the end;
[0081] Adding a linker to the free nucleic acid with "A" added to the end to obtain a linker-added free nucleic acid;
[0082] mixing the plurality of nucleic acid fragments containing the tag sequence of the sample;
[0083] Amplifying the adapter-added free nucleic acid mixture to obtain an enriched product of the adapter-added free nucleic acid, and recovering the enriched product with magnetic beads to obtain a nucleic acid sequencing library.
[0084] The nucleic acid sequencing library is screened for a predetermined range of nucleic acid fragments to obtain a predetermined range of nucleic acid sequencing libraries, and the screening methods...
Embodiment 2
[0098] Two analyzes were performed on 6 samples. That is to say, two libraries were constructed from the same sample at the same time, and one of the libraries was screened for target fragments using agarose gel, and the target fragments in the range of 130-150bp were screened out. The other library was not screened on agarose gel and was sequenced on the machine at the same time to determine whether the library that had been screened by fragments contained a higher fetal concentration than the library that had not been screened by fragments.
[0099] 1. Sample collection and processing
[0100] Use Streck blood collection tubes to collect 5-10mL of peripheral blood from pregnant women. After collection, plasma separation is performed according to the standard two-step centrifugation method.
[0101] 2. Extraction of free DNA from peripheral blood plasma of pregnant women
[0102] The TIANamp Micro DNA Kit was used to extract the free DNA from the peripheral plasma of pregnant...
Embodiment 3
[0176] The method of this example is basically the same as that of Example 2, the difference being that magnetic beads are used to screen the target fragment. The steps of the magnetic bead screening method are as follows:
[0177] The sample to be selected was transferred to a 1.5mL centrifuge tube, and the amplified sample was purified with AMPure XP DNA Purification kit (SPRIbeads).
[0178] 1. Take out the Ampure XP Beads stored at 4°C, and place them at room temperature for 30 minutes to equilibrate;
[0179] 2. Shake evenly before use, add magnetic beads according to the volume of 0.8-0.9 times the sample volume and mix well, let stand for 2 minutes, and centrifuge for 3 seconds instantaneously.
[0180] 3. Transfer the 1.5mL centrifuge tube to the magnetic stand and let it stand for 3-5min until clarified;
[0181] 4. Carefully suck off the supernatant, transfer to a new 1.5ml centrifuge tube, and discard the original centrifuge tube;
[0182] 5. Add 0.3-0.4 times ma...
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