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DNA sample preparation method suitable for trace sample

A sample and micro-volume technology, applied in biochemical equipment and methods, micro-organism measurement/inspection, etc., can solve the problem that the CTAB method is not suitable for micro-sample sequencing, and achieve the effect of simple operation

Active Publication Date: 2022-07-01
SHENZHEN HUADA GENE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At least in order to solve the problem that the CTAB method commonly used in the sequencing of plant whole genome libraries is not suitable for micro-sample sequencing, the present invention proposes a new DNA preparation method suitable for micro-sample, which is especially suitable for micro-scale plant or algae whole-body sequencing. genome sequencing

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  • DNA sample preparation method suitable for trace sample
  • DNA sample preparation method suitable for trace sample
  • DNA sample preparation method suitable for trace sample

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Embodiment 1

[0031] 1. DNA sample preparation

[0032] a) Sample preparation for micro-plant DNA-free extraction (magnetic bead method)

[0033]Take a cotyledon or leaf (about 1 mg) with an area of ​​about 0.2 cm × 0.2 cm and place it in 20 μL of 1% SDS solution, mash the tissue with a stirring rod (10 s), place it in liquid nitrogen for 3 min, and then quickly put it into a 60°C water bath 7min. Following centrifugation at 12000 rpm for 3 min, the supernatant was transferred to a new centrifuge tube. The supernatant was adjusted to 50 μL with TE, 1.5× (75 μL) AmPureXP Beads magnetic beads were added to purify the diluted product, and the magnetic beads were equilibrated at room temperature for 30 min. Gently mix the supernatant and magnetic beads in the centrifuge tube for 10 times, incubate at room temperature for 10 min, and then place on a magnetic stand for adsorption for 5 min. After the liquid is clarified, aspirate and discard the supernatant. Add 200 μL of 80% ethanol without d...

Embodiment 2

[0129] Whole Genome Sequencing of Chlorella microflora without DNA Extraction and Purification

[0130] In order to explore a more suitable concentration of lithium salicylate, Example 1 uses a final concentration of 2.5mM lithium salicylate for DNA-free extraction and purification of trace algae, while this example uses a final concentration of 0.5, 1.5, 3.5 and 5mM salicylates in parallel. Lithium oxide was used to treat the same microalgae samples. details as follows:

[0131] will 10 4 Chlorella cells were treated with liquid nitrogen at final concentrations of 0.5, 1.5, 3.5 and 5 mM C 7 H 5 LiO 3 Process for 10min. After the algae liquid was centrifuged at 12000rpm for 3min, 2ng DNA of the supernatant was directly interrupted with 0.3μL Tn5 for 5min, and then the adapter primer with barcode was used to amplify and recover the amplified fragment (for specific steps, see Example 1 using salicylic acid). Lithium method for microalgal sample preparation for DNA-free ext...

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Abstract

The invention belongs to the technical field of biology, and discloses a DNA sample preparation method suitable for a trace sample, and the method comprises the following steps: 1) treating the trace sample with liquid nitrogen; (2) raising the temperature of the sample treated by the liquid nitrogen, and incubating; and 3) treating the incubated sample with lithium salicylate or purifying the incubated sample with magnetic beads to obtain the DNA sample of the trace sample. According to the method disclosed by the invention, enough DNA required for sequencing and library building can be extracted only by using the order of magnitude as low as 1mg or 104 cells, the DNA extracted by using the method disclosed by the invention has fewer impurities, and the input amount of the DNA for sequencing and library building is as low as 2ng.

Description

technical field [0001] The present invention belongs to the field of biotechnology, and more specifically, the present invention provides a DNA sample preparation method suitable for trace samples, especially trace samples of plants or algae. Background technique [0002] Obtaining sufficient DNA samples for whole-genome sequencing of microscopic plants and algae is a challenge due to their relatively small sample sizes. Existing plant whole-genome library sequencing usually requires first using CTAB and other methods to extract whole-genome DNA from plant tissue, and then using whole-gene shotgun method to interrupt and amplify DNA fragments to establish a sequencing library. The extraction of whole genome DNA by CTAB usually requires at least 100 mg of plant tissue samples, so it takes a long time for the growth of trace plants or algae to obtain enough samples. Using the CTAB method to extract whole genome DNA, plant samples need to be ground in liquid nitrogen and extra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806
CPCC12Q1/6806C12Q2563/143C12Q2563/149
Inventor 顾颖刘光宇夏科科杨勇
Owner SHENZHEN HUADA GENE INST