Method for detecting X-chromosome fragile molecular sequence by using nanopore sequencing technology

A nanopore sequencing and X chromosome technology, applied in the field of biomedicine, can solve the problems of complex technology, short sequencing read length, unable to detect repeat sequence length, etc., and achieve the effect of simple operation and sensitive results in the detection process

Inactive Publication Date: 2015-07-01
代苒
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the sequencing read length of traditional Sanger sequencing and next-generation sequencing technology is relatively short (about 300-1000 bases), and it is difficult for Taq DNA polymerase to amplify short repeat sequences, the CGG repeat sequence of the FMR1 gene cannot be effectively detected
At present, the detection of the CGG repeat sequence of the FMR1 gene relies on Southern (Southern) blot hybridization and several specially designed polymerase chain reaction (PCR) techniques. The standard of the laboratory is very high, and the accuracy of the test is not satisfactory
In addition, the indirect detection of transcriptional repression of the promoter of the FMR1 gene using methylation-PCR technology cannot detect the length of the repeat sequence

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Example 1: Step 1: FMR1 gene enrichment.

[0013] As a preference, heat treatment is used to carry out fragmentation pretreatment of DNA:

[0014] 1-2 micrograms of DNA samples (volume 50 microliters) were heat-treated in a PCR cycler (90°C for 30 minutes, 4°C ∞). Centrifuge briefly. The size of DNA fragments can be determined by agarose gel electrophoresis. The fragmented DNA was then end-repaired using the Next End-Repair module kit. After end repair, the blunt-ended DNA was purified using an equal volume of AMPure XP beads to remove unreacted reagents and substrates. End modification was performed using the Next dA-tailing module kit with a final reaction volume of 30 µl.

[0015] The enrichment of the target gene can be achieved by liquid phase sequence capture technology (In solution capture), that is, designing and synthesizing a single-stranded oligonucleotide probe (with biotin label) targeting the homologous segment of the FMR1 gene. The DNA fragments are ...

Embodiment 2

[0019] Example 2: 1. Enrichment of FMR1 gene: Using long range PCR, design specific PCR primers on both sides of the gene segment containing FMR1, through special DNA polymerase and special reaction The program directly amplifies the segment containing the gene of interest.

[0020] Long range amplification was performed using the Long Range Hot Start PCR system kit. The specific experimental conditions and reaction system are as follows: 5× KAPA Long Range Buffer (without Mg 2+ ) 1×, MgCl 2 (25 mM) 1.75 mM, dNTPs (10 mM each dNTP) 0.3 mM, Fwd primer (10 μM) 0.5 μM, Rev primer (10 μM) 0.5 μM, 50ng DNA, KAPA Long Range Hot Start DNA Polymerase (2.5 units / micro L) 1.25 units / 50 µl for a total volume of 50 µl.

[0021] Primer sequences used: FMR1-Forward (TCA GGC GCT CAG CTC CGT TTC GGT TTC A), FMR1-Reverse (AAG CGC CAT TGG AGC CCC GCA CTT CC).PCR reaction conditions: 98°C for 5 min; 25 cycles of 97 °C 35 s, 62°C 35 s, and 72°C 4 min; a final extension at 72°C 10 min.

[00...

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PUM

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Abstract

A nanopore sequencing technology is applied to detecting an X-chromosome fragile molecular sequence for the first time in the invention. A method for detecting the X-chromosome fragile molecular sequence by using the nanopore sequencing technology comprises the following steps: enriching FMR1 genes; sequencing nanopores; and analyzing the sequence. By means of the nanopore sequencing technology, the sequencing process of the X-chromosome fragile molecular sequence is simpler to operate, and the result is more sensitive.

Description

technical field [0001] The present invention relates to biomedicine, in particular to gene sequencing steps of biomedicine. Background technique [0002] Fragile X is the second leading cause of inherited intellectual disability after Down syndrome and one of the causes of autism. In addition to intellectual disability, common symptoms include the patient's malocclusion, hypotonia, and idiosyncratic facial features. [0003] It is known that the causative gene of Fragile X is the FMR1 gene located on the X chromosome, and the gene silencing caused by the over-expansion of the CGG repeat sequence in the untranslated region of the 5' end of the gene is the known main cause. Effective detection of the length of the CGG repeat sequence of the FMR1 gene is of great practical significance for the diagnosis of Fragile X chromosome and prenatal detection. Because the sequencing read length of traditional Sanger sequencing and next-generation sequencing technology is relatively sho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q2525/151C12Q2531/113C12Q2565/631
Inventor 代苒
Owner 代苒
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