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Signal combination coding-based DNA ligation sequencing method

A DNA sequencing and signal combination technology, applied in the biological field, can solve the problems of single signal labeling method, low efficiency, sequencing cost, throughput and speed that cannot meet the needs of life sciences, etc.

Active Publication Date: 2009-11-04
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current next-generation DNA sequencing still cannot meet the needs of life science research in terms of sequencing cost, throughput and speed.
One of the main reasons is that the signal labeling method for detecting nucleic acid is single and the efficiency is not high.

Method used

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  • Signal combination coding-based DNA ligation sequencing method
  • Signal combination coding-based DNA ligation sequencing method
  • Signal combination coding-based DNA ligation sequencing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: The present invention is a DNA ligation sequencing method based on signal combination coding, which is characterized in that a specific DNA sequencing probe is labeled with a combination of multiple marker states, and a batch of DNA sequencing probes are used for labeling. The needle is labeled with different combinations of multiple marker states to prepare a set of DNA sequencing probes encoded by the signal combination, so as to distinguish and identify different DNA sequencing probes during detection, so as to achieve the goal of sequencing the template to be tested. purpose.

[0048]A. Preparation of DNA sequencing probes encoded by a set of signal combinations: first prepare unlabeled DNA sequencing probes. Each unlabeled DNA sequencing probe consists of one or more sequencing bases and one or more degenerate bases. Base N or non-strictly paired base composition. Sequencing bases are used to determine the base information of the corresponding position in th...

Embodiment 2

[0065] Example 2: Four-color fluorescence combination coding method (respective labeling scheme) detects a total of 32 base pair sequences of 45332-45363 on human chromosome 17 on the RP11-354P11 clone;

[0066] Extract human whole genome DNA, use PCR method to amplify the target fragment, the primers are P1, P2 (see Table 3 for the sequence), and the 5'end of primer P1 is modified with hydroxyl group. After the amplified PCR product is fixed on the surface of the aldehyde-modified glass slide, the glass slide is heated to 95°C to separate one strand of the double-stranded PCR product from the fixed strand, and then the sequencing reaction is started.

[0067] Table 3 Related oligonucleotide sequence information in Example 2

[0068] Sequence name

Sequence information

Sequence to be tested

5’-CACGGACCAGCTGCCCTGGACCAGCTGCAAGA-3’

P1

OH-5’-CGCTATACTACCTCATCTCCTCCTTCACG-3’

P2

5’-GCAGTTGCCAGTGTTCCAGGAGT-3’

I1

5’-GTTCCAGGAGT...

Embodiment 3

[0077] Example 3: Four-color fluorescence combination coding method (common labeling scheme) detects a total of 32 base pair sequences of 45332-45363 on human chromosome 17 on the RP11-354P11 clone;

[0078] Extract human whole genome DNA, use PCR method to amplify the target fragment, the primers are P1, P2 (see Table 5 for the sequence), and the 5'end of primer P1 is modified with hydroxyl group. After the amplified PCR product is fixed on the surface of the aldehyde-modified glass slide, the glass slide is heated to 95°C to separate one strand of the double-stranded PCR product from the fixed strand, and then the sequencing reaction is started.

[0079] Table 5 Related oligonucleotide sequence information in Example 3

[0080] Sequence name

Sequence information

Sequence to be tested

5’-CACGGACCAGCTGCCCTGGACCAGCTGCAAGA-3’

P1

OH-5’-CGCTATACTACCTCATCTCCTCCTTCACG-3’

P2

5’-GCAGTTGCCAGTGTTCCAGGAGT-3’

I1

5’-GTTCCAGGAGTNNNN...

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Abstract

The invention discloses a signal combination coding-based DNA ligation sequencing method, which relates to a DNA ligation sequencing method using signal combination coding and belongs to the technical field of biology. The method is characterized by connecting signal codes in a ligation sequencing reaction, realizing the sequencing process of synchronously detecting base types in a number which is a power of the number of markers in one ligation reaction by using the same number of markers, exponentially reducing sequencing time, considerably improving sequencing throughput and reducing sequencing cost. The method uses the combinations of the signal statuses in the detection of the prior a plurality of markers for coding, makes the combinations correspond to the types of detected bases one by one and improves the resolution power for markers in the same number due to the fact that the number of the combinations of the signal statuses of the plurality of markers exponentially grows with the increase of the number of the types of the markers, thereby detecting the base types in the number which is the power of the number of the markers in one sequencing reaction.

Description

Technical field [0001] The DNA connection sequencing method based on signal combination coding relates to a DNA connection sequencing method adopting signal combination coding, is a method for realizing DNA sequence analysis, and belongs to the field of biotechnology. technical background [0002] With the deepening of genome research, especially the completion of the Human Genome Project and various model organism genome projects, the methods of biological research and medical research have undergone tremendous changes. It is possible to understand the differences in life from the genetic level, the law of occurrence and development of diseases, the interaction between drugs and living organisms, the genetic differences between different species, and the genetic differences between different individuals within the same species. Although there are many factors that cause disease, genetic sequence differences (including single nucleotide polymorphisms, DNA methylation, etc.) are w...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 涂景陆祖宏白云飞葛芹玉孙蓓丽
Owner SOUTHEAST UNIV
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