Multiple detection method for DNA polymorphism of genome and special probe thereof
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A polymorphism, genome technology, applied in the field of genetic engineering, can solve the problem of low throughput
Inactive Publication Date: 2011-09-14
INST OF BOTANY CHINESE ACAD OF SCI
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The traditional method for detecting insertion / deletion markers is mainly to design a pair of PCR primers for an insertion / deletion site, and through PCR amplification, gel or capillary electrophoresis detection, generally only one insertion / deletion marker can be detected at a time, and the throughput is very high. Low
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Embodiment 1
[0110] Embodiment 1, the button hand probe of single nucleotide polymorphism (SNP)
[0111] 1. Detection of two SNPs in the genome of Aegilops tauschii
[0112] 1. Design of button probes PdPCJ819266A, PdPCJ819266G, PdPBJ262615T, PdPBJ262615C
[0113] The genome of Aegilops tauschii is the donor of the D genome of common wheat (Triticum aestivum). PdPCJ819266 and PdPBJ262615 are two SNPs of Aegilops tauschii (Aegilops tauschii), in which AA and TT are genotypes of Aegilops tauschii variety 2280, and GG and CC are genotypes of Aegilops tauschii variety 2282, that is, Aegilops tauschii 2280 and Aegilops tauschii The 62nd nucleotide in the sequence 5 of the 2282 variety has a polymorphism, which are AA and GG respectively; the 175th nucleotide in the sequence 6 of the 2280 and 2282 varieties of A. tachyphylla has a polymorphism, which are respectively TT and CC.
[0114] Among them, the button-hand probe PdPCJ819266A specifically recognizes allele A with a length of 147 nt, th...
[0162] 1. Detection of two insertion / deletion (InDel) markers in the barley genome
[0163] 1. Design of button probes PdPInDel1-1, PdPInDel1-2, PdPInDel2-1, PdPInDel2-2
[0164] PdPInDel1 and PdPInDel2 are two InDel markers on the fifth chromosome (5H) of barley (Hordeum vulgare L.), wherein InDel1-1 and InDel2-1 are specific genotypes of barley variety Vada, and InDel1-2 and InDel2-2 are Genotypes specific to barley cultivar L94. The button-hand probe PdPInDel1-1 specifically recognizes the InDel1-1 genotype, and its length is 160 nt. The button-hand probe PdPInDel1-2 specifically recognizes the InDel1-2 genotype, and its length is 190 nt. The genotype is 318nt in length, and the clasp probe PdPInDel2-2 specifically recognizes the InDel2-2 genotype with a length of 342nt.
[0165] The 5' ends of the four hand probes were all phosphorylated. See Table 4 for sequence design and structur...
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Abstract
The invention discloses a multiple detection method for deoxyribose nucleic acid (DNA) of a genome and a special probe thereof, and provides a single chain DNA molecule. The single chain DNA molecule consists of the following segments sequentially from 5' tail end to 3' tail end: a left side oligonucleotide segment complementary with a 5' end of DNA to be detected, a main body segment and a rightside oligonucleotide segment complementary with a 3' end of the DNA to be detected. Experiments prove that: in the single chain DNA molecule provided by the invention, loci with different markers andalleles are distinguished from one another through a ligase reaction, buckled probes which have different lengths and are successfully connected through polymerase chain reaction (PCR) amplification can simultaneously detect the polymorphism of a plurality of DNA markers through a gel or capillary electrophoretic separation and detection system, high sensitivity and accuracy can be realized, and the requirements of high throughout and low cost are met simultaneously.
Description
technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for multiple detection of genomic DNA polymorphism and a special probe thereof. Background technique [0002] Deoxyribonucleic acid (DNA) polymorphism refers to the difference in nucleotide arrangement in chromosomal DNA alleles, that is, there are two or more genotypes in the alleles (or fragments) in the DNA region, which can be divided into mononuclear Nucleotide polymorphism (single nucleotide polymorphism, SNP) and insertion / deletion polymorphism (insertion / deletion, In / Del). [0003] (1) Single Nucleotide Polymorphism (SNP) [0004] Single nucleotide polymorphism (SNP) refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the chromosome genome level, in which at least one allele occurs in no less than 1% of the population. The polymorphism shown by SNP only involves the variation of a single base, which can be caused by the t...
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