WO2011120409A1 - 以alu间聚合酶链式反应为基础的检测基因区特征的方法 - Google Patents

以alu间聚合酶链式反应为基础的检测基因区特征的方法 Download PDF

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WO2011120409A1
WO2011120409A1 PCT/CN2011/072204 CN2011072204W WO2011120409A1 WO 2011120409 A1 WO2011120409 A1 WO 2011120409A1 CN 2011072204 W CN2011072204 W CN 2011072204W WO 2011120409 A1 WO2011120409 A1 WO 2011120409A1
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alu
dna
sequence
inter
aluy
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PCT/CN2011/072204
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薛红
梅玲玲
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广州市香港科大霍英东研究院
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Priority to US13/637,444 priority Critical patent/US20130040828A1/en
Publication of WO2011120409A1 publication Critical patent/WO2011120409A1/zh
Priority to US13/678,693 priority patent/US20130143746A1/en

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    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the invention belongs to the field of biotechnology. Specifically, it relates to a method for collectively detecting single nucleotide polymorphism (SNP), point mutation, sequence insertion/deletion, and dideoxynucleotide (DNA) methylation CpG site in a gene region.
  • the method uses the Alu family, especially the AluY subfamily common sequence as the main oligonucleotide primer, and uses a plurality of primers Alu to amplify genomic DNA by polymerase chain reaction (inter-Alu PCR), and amplifies the DNA replicon in a single time. Most focus on each gene region in the genome.
  • the method is characterized in that part of the non-gene region sequence can be filtered by inter-Alu PCR, so that the next generation sequencing technology can focus on detecting SNPs, point mutations, sequence insertion/deletion and DNA methylation CpG sites in the gene region of the genome. It also saves the amount of genomic DNA required for sequencing.
  • This advancement in biotechnology has greatly reduced the cost of whole-genome sequencing and has greatly accelerated the speed of disease etiology research.
  • This technology is currently used in the correlation study of various diseases including tumors and mental diseases.
  • 3 ⁇ ⁇ genomic DNA is required, and 3 ⁇ ⁇ usually does not meet the requirements for whole genome sequencing.
  • the need for a high total amount of DNA reduces the acceptance of the sample provider.
  • the data from a single sequencing may focus only on certain areas of the genome, losing other important areas needed for the study.
  • PCR Polymerase chain reaction
  • U.S. Patent Nos. 5,773,649, 6, 060, 243 and 7, 537, 889 all relate to the use of Alu inter-Alu PCR to amplify multiple regions of human genomic DNA.
  • patent 5, 773, 649 is the use of inter-Alu PCR The technique amplifies tumor genomic DNA and peripheral blood DNA of the same patient, and observes the tumor genomic DNA replication error phenotype by DNA agarose gel electrophoresis.
  • specific mutations in the genome of the disease have not been studied in depth.
  • AluY subfamily acts as a recombination hotspot, and SNPs within and around the structure may be disease-associated SNPs.
  • the Alu family can contain up to 33% of CpG sites throughout the human genome. Previous studies have shown that in the tumor genome, the methylation level of some specific CpG sites in the Alu sequence is significantly reduced, which is particularly prominent in the AluY subfamily.
  • the inter-Alu PCR using the AluY consensus sequence as an oligonucleotide primer can achieve detection of SNPs, point mutations, insertion/deletion, and methylation level of DNA CpG sites in the gene region.
  • This improved method allows for the use of trace amounts of DNA in disease etiology studies to obtain more efficient sequencing data from high-throughput sequencing instruments, meeting the cost-quality-quantity balance requirements of the study.
  • the present invention relates to a method for collectively detecting a single nucleotide polymorphism (SNP), a point mutation, an insertion/deletion, and a dideoxynucleotide (DNA) methylation CpG site in a gene region.
  • this method is a method that enables next-generation sequencing technology to centrally detect the sequence characteristics of gene regions in the genome through inter-Alu PCR.
  • the multi-primer inter-Alu PCR amplification of the region between the two Alu sequences in the genome, the quality of the purified PCR product is 6 times the mass of the template genomic DNA; and, the PCR The product is a DNA replicon that encompasses multiple gene regions in the genome.
  • This approach ensures that next-generation sequencing technologies use only nanogram genomic DNA to centrally detect SNPs, point mutations, sequence insertions/deletions, and DNA methylation CpG sites in the gene region.
  • the oligonucleotide primer design is based on the characteristics of AluY described above, and the fragments in the common sequence of the AluY subfamily are selected as oligonucleotide primers.
  • the AluY common sequence small fragment oligonucleotide primer can be complementary to the detected template DNA at the appropriate sequence position by the action of the thermostable DNA polymerase.
  • a hybrid nucleic acid structure is formed.
  • the oligonucleotide primers are extended with four nucleotides (A, G, T, C), and the base is complementary to the thermostable DNA.
  • the template chain to be amplified is catalyzed by a polymerase to achieve the purpose of replicating the template strand.
  • the replication of DNA is exponentially amplified, that is, the quality of the PCR product is greater than the quality of the original template DNA.
  • the quality of the purified PCR product is 6 times the mass of the original template.
  • the form of the amplified product was observed to be a white stepped shadow under an ultraviolet lamp by ethidium bromide staining. If there are a plurality of small fragment oligonucleotide primers in the PCR reaction, the amplified product form under ultraviolet light is a white patchy shadow.
  • the DNA replicon amplified by the AluY common sequence small fragment oligonucleotide primer inter-Alu PCR is 40% in the gene region. The ratio of the entire gene region to the entire genome is only 25%.
  • the inter-Alu PCR product amplified by the 3 ⁇ ⁇ ng-type DNA template can be sequenced in a high-throughput sequencing instrument, and the resulting multi-site SNPs are mainly concentrated in the Alu fragment of the gene region and the fragment around Alu. in. Because about 50% of the SNPs are distributed in the repeat region, the rest are distributed in non-coding sequence regions and coding gene regions outside the repeat sequence. Therefore, the method can be used to detect multiple SNPs in the genome, particularly in the gene region, and to discover new SNPs.
  • Another embodiment of the present invention is the use of the above method for detecting point mutations, insertions/deletions of various diseases, particularly gene and introns in the genome of the cancer.
  • the number of genes in the entire genome is about 25,000, and the number of tumor-related genes is now known to be 6522, accounting for 26% of the total number of genes.
  • tumor genomic DNA is amplified by inter-Alu PCR using a small fragment of the AluY common sequence as an oligonucleotide primer, and 58% of the gene gene sequence is a tumor-associated gene.
  • Alu-common sequence primers (R12A/267) with oligonucleotide primers and "tail-to-tail" amplification direction including AluY common sequence with different amplification directions can be used as mixed primers in inter-Alu
  • a multi-oligonucleotide primer is complementary to a template DNA to be detected at an appropriate sequence position by the action of a thermostable DNA polymerase to form a hybridized nucleic acid structure.
  • the oligonucleotide primers are extended with four nucleotides (A, G, T, C), and the base-complementing principle is catalyzed by a thermostable DNA polymerase and amplified.
  • the template chain is connected to achieve the purpose of copying the template chain. Since a plurality of primers acted together in the same PCR reaction system, the obtained DNA product was subjected to DNA agarose gel electrophoresis, and stained with ethidium bromide, and the form of the amplified product was observed as a white patchy shadow under ultraviolet light observation. According to the technique described above, it is possible to detect whether the SNP contained in the inter-Alu PCR amplification region is a tumor phase The related SNPs can be further disrupted by the DNA replicon. Combined with the exon capture technique, the correlation between the SNPs in the exons of the amplified region and the tumor is analyzed.
  • Another embodiment of the invention is the use of the above method for the detection of the level of DNA methylation in the gene region.
  • Cytosine (C) methylation in DNA occurs mostly in 5'-CpG-3' "C” ⁇ , resulting in 5-methylcytosine (5mC).
  • 5mC 5-methylcytosine
  • the vast majority of 5mC is mainly concentrated in the Alu family of repetitive sequences rich in CpG sites, and it is estimated that the Alu family can contain up to 33% of CpG sites throughout the human genome. Therefore, two oligonucleotides with different amplification directions were designed with a sequence containing no CpG site in the AluY common sequence, and all "C" in the primer was converted into "T”.
  • PCR products of Alu and its surrounding regions rich in CpG sites in the genome can be obtained.
  • Two oligonucleotide primers with different amplification directions work together on the same inter-Alu PCR amplification reaction system, which can include "tail-to-tail”, “head-to-head”, “head-to-tail” and "tail-to-head”
  • the four-way DNA replicon further increases the number of CpG locus regions, allowing the sequence obtained by sequencing by next-generation sequencing technology to provide as much information as possible of the degree of methylation level of the CpG locus in the tumor genome.
  • gene region refers to the region in which the gene is located in the genome sequence.
  • a gene is the material basis of heredity. It is a general term for a specific nucleotide sequence with genetic information on a DNA molecule, and is a fragment of a DNA molecule with a genetic effect.
  • purified PCR product refers to the removal of excess primers, enzymes, mineral oil, glycerol, salt and other impurities after passing the inter-Alu PCR reaction by removing the PCR product from ethanol or a purification kit. Purified.
  • region between two Alu sequences refers to the region in which the inter-Alu PCR amplification product is located. Since the Alu sequence is widely distributed in the genome, the DNA sequence between the adjacent two Alu sequences included is amplified under the tolerance of the DNA length that the thermostable DNA polymerase can amplify.
  • quality means two aspects. First, refers to the number of PCR products amplified in inter-Alu. Second, the extent to which the sequencing data meets the research requirements.
  • DNA replicon refers to an inter-Alu PCR amplification product, here The PCR product obtained by amplifying genomic DNA with Alu oligonucleotide primers.
  • next generation sequencing technology refers to a more advanced circular array synthesis sequencing method than the first generation sequencing technology (fluorescent-labeled Sanger method). This technique allows for the acquisition of sequences of several million bases in a single sequence.
  • ng no-level genomic DNA
  • AluY common sequence small fragment oligonucleotide primer as used in the present specification means that 10-20 bases in the common sequence of the AluY subfamily are selected as primers and used in the inter-Alu PCR reaction. Fragments of these primers can be selected from any position in the AluY common sequence as required by different experiments.
  • white stepped shadow refers to a DNA replicon produced by an inter-Alu PCR reaction of single or multiple oligonucleotide primers, which is subjected to DNA agarose gel electrophoresis and stained with ethidium bromide. The form shown under the UV lamp. Its color is white and its shape is like a ladder.
  • white patchy shadow refers to a DNA replicon produced by an inter-Alu PCR reaction of a plurality of oligonucleotide primers, which is subjected to DNA agarose gel electrophoresis and stained with ethidium bromide. The form shown under the UV lamp. Its color is white, shape cloud or patch.
  • thermostable DNA polymerase refers to a DNA polymerase used in an inter-PCR reaction.
  • the thermostable DNA polymerase may be a Taq enzyme, a KOD enzyme or various other DNA polymerases which can be used in various PCR reactions.
  • different amplification direction means that the Alu has a "5,” end head and a “3'” end tail, and the amplification direction of the inter-Alu PCR may be toward the "5'” end, also Can be oriented towards the "3'” end. Therefore, inter-Alu PCR amplification can occur in different directions.
  • tail-to-tail means that the Alu has a "5'” end and a “3'” end, inter-Alu PCR amplifies an Alu “3'” end with an Alu "3' "The DNA region between the ends of the tail.
  • head-to-head means that the Alu has a "5'” end and a “3'” end, inter-Alu PCR amplifies an Alu “5,” end head with an Alu "5, " The DNA region between the ends of the head.
  • head-to-tail means that the Alu has a "5'” end and a "3'” end, inter-Alu PCR amplifies an Alu "5'” end with a Alu "3 '”End tail The DNA region between.
  • tail-to-head means that the Alu has a "5'” end and a “3'” end, and inter-Alu PCR amplifies an Alu "3'” end with an Alu” 5, "The DNA region between the ends of the head.
  • exon capture refers to the construction of a vector from which an exon sequence is recognized and recovered, thereby cloning the gene of interest.
  • the inter-Alu PCR product is mainly used as a template for exon capture.
  • CpG site means that cytosine (C) in DNA forms a linear linkage with guanine (G) via a phosphodiester bond.
  • the level of "C” methylation in the CpG site in mammals is 70% to 80%.
  • the present invention relates to a method of collectively detecting the characteristics of a gene region.
  • This method is a method of concentrating the sequence of the gene region in the genome by inter-Alu PCR.
  • a high quality purified PCR product can be obtained by amplifying a plurality of two Alu sequence regions in the genome; and the PCR product covers multiple gene regions in the genome. DNA replicon.
  • This method ensures that the next-generation sequencing technology uses only nanogram genomic DNA to achieve the purpose of centrally detecting the characteristics of the gene region.
  • the AluY subfamily is inserted into the genome to cause genomic instability, and in addition, the subfamily acts as a recombination hotspot, and the SNPs inside and around the structure may be disease-related. SNP; Therefore, the design of the oligonucleotide primers is based on the above characteristics of AluY, and the fragments in the common sequence of the AluY subfamily are selected as oligonucleotide primers.
  • the AluY common sequence small fragment oligonucleotide primer can complement the detected template DNA at an appropriate sequence position by the action of the thermostable DNA polymerase to form a hybrid nucleic acid structure. Then, under this polymerization condition, the oligonucleotide primers are extended with four nucleotides (A, G, T, C), and the base-complementing principle is catalyzed by a thermostable DNA polymerase and amplified.
  • the template chain is connected to achieve the purpose of copying the template chain. According to the amplification characteristics of PCR, the replication of DNA is exponentially amplified, that is, the quality of the PCR product is greater than the quality of the original template DNA.
  • the purified PCR product has a mass of 6 times that of the original template.
  • a single AluY common sequence small fragment oligonucleotide primer can amplify multiple regions in the genome by inter-Alu PCR to obtain multiple sizes.
  • Different DNA complex System After DNA agarose gel electrophoresis, the form of the amplified product was observed as a white stepped image by ultraviolet light irradiation. If there are a plurality of small fragment oligonucleotide primers in the PCR reaction, the amplified product form under ultraviolet light is a white patchy shadow.
  • the DNA replicon amplified by the AluY common sequence small fragment oligonucleotide primer inter-Alu PCR is 40% in the gene region.
  • the ratio of the entire gene region to the entire genome is only 25%.
  • Another embodiment of the present invention is the use of the above method for detecting point mutations, insertions/deletions of different diseases, particularly gene and introns in the tumor genome.
  • the number of genes in the entire genome is about 25,000, and the number of tumor-related genes is currently known to be 6522, accounting for 26% of the total number of genes.
  • tumor genomic DNA is amplified by inter-Alu PCR using a small fragment of the AluY common sequence as an oligonucleotide primer, and 58% of the gene gene sequence is a tumor-associated gene.
  • an AluY common sequence or an Alu common sequence including different amplification directions can be used as an oligonucleotide primer, and in an inter-Alu PCR, a plurality of oligonucleotide primer mixtures are used to thermally stabilize a DNA polymerase.
  • the multiple oligonucleotide primers are complementary to the template DNA being detected at the appropriate sequence positions to form a hybridized nucleic acid structure. Then, under this polymerization condition, the oligonucleotide primers are extended with four nucleotides (A, G, T, C), and the base-complementing principle is catalyzed by a thermostable DNA polymerase and amplified.
  • the template chain is connected to achieve the purpose of copying the template chain.
  • the obtained DNA product was subjected to DNA agarose gel electrophoresis, and stained with ethidium bromide, and the form of the amplified product was observed as a white patchy shadow on an ultraviolet lamp.
  • disease-associated SNPs, insertions/deletions in the inter-Alu PCR amplification region can be detected.
  • the DNA replicon can be further interrupted, and the exon capture technology is combined to analyze the correlation between SNP and tumor in the exon of the amplified region.
  • Another embodiment of the invention is the use of the above method for the detection of the level of DNA methylation in the gene region.
  • two amplification oligonucleotide primers with different amplification directions were designed with the sequence of the AluY-free sequence without CpG site, and the "C” in the primer was all Convert to "T”.
  • the bisulfite pretreated genomic DNA was amplified by inter-Alu PCR, and PCR products of multiple sites in the genome, CpG-rich Alu and its surrounding regions were obtained.
  • Two different orientation amplification primers work together on the same inter-Alu PCR amplification reaction system, which can be obtained by including the "tail pair"
  • the DNA replicons in the four directions of tail, head-to-tail, and tail-to-head can provide the sequence of methylation of genomic CpG sites as much as possible by sequencing sequences obtained by sequencing of next-generation sequencing technologies.
  • This method can be used to detect the level of methylation in normal human genomes or to analyze the extent of changes in DNA methylation levels in various disease states.
  • Figure 1 is a schematic illustration of SNPs in the gene region in Example 1.
  • the amplified fragment is mainly located in the gene region, and a SNP is included in the In the gene region.
  • Figure 2 is a diagram showing the positions of two oligonucleotide primers designed by the inter-Alu PCR in Example 1 in a small fragment of the AluY consensus sequence in the AluY structure, and the amplification directions of the two primers in the genomic DNA. schematic diagram. Because AluY has a "5'" end and a "3'” end, and their distribution is not uniform across the genome, two adjacent Alus at appropriate distances can be amplified by inter-Alu PCR. come out. This results in amplification as shown in Figure 2, toward the "5'" direction and toward the "3'” direction.
  • Figure 3 is a schematic illustration of the specific positions and sequences of two AluY co-sequence oligonucleotide primers in Example 1 in AluY.
  • the oligonucleotide primer (AluT-T) amplified in the "tail-to-tail” direction is located at positions 182 to 200 of the AluY common sequence, and the base is "5, -AGGCTGAGGCAGGAGAATG-3, : “head to head”
  • the direction-amplified oligonucleotide primer (AluH-H) is located at position 66 of the AluY common sequence to position 86 of IJ, and the base is "5, -TGGTCTCGATCTCCTGACCTC-3 ', .
  • Figure 4 is a photograph (inverse color display) of the amplified DNA replicon in the agarose gel observed in the ultraviolet lamp of Example 1.
  • On the left is the image of the tail-to-tail AluY oligonucleotide primer inter-Alu PCR amplification product in an agarose gel, such as a stepped shape; the middle is the DNA molecular marker (lkb) (produced by Fermentas); An image of the "head-to-head" AluY oligonucleotide primer inter-Alu PCR amplification product in an agarose gel, such as a step.
  • the bands amplified by the inter-Alu PCR of the two oligonucleotide primers were mainly between 300 bp and 2 kb.
  • the arrow refers to the strip position where the gel is sequenced.
  • Figure 5 is a schematic diagram showing the sequence of the oligonucleotide primer (AluYTl) near the tail of the AluY structure schematic diagram and the position in AluY in Example 2.
  • the positions of the oligonucleotide primers range from position 278 to position 295 in the AluY consensus sequence.
  • the base is "5'-GAGCGAGACTCCGTCTCA-3'". It can Used to display tumor genomic DNA replication error phenotypes.
  • Figure 6 is a schematic illustration of the amplification direction of three oligonucleotide primers.
  • an oligonucleotide primer of AluY common sequence and Alu common sequence oligonucleotide primer R12A/267 can amplify between one Alu "3 '" end and another Alu "3 '” end.
  • DNA sequence (“tail-to-tail” orientation)
  • another AluY consensus sequence oligonucleotide primer can amplify a DNA sequence between the Alu "5'" end and another Alu "5'” end (" Head to head "direction”.
  • Mixed oligonucleotide primers can also amplify a DNA sequence between the Alu “5'” end and another Alu “3'” end ("head-to-tail” orientation), and an Alu “3'” end with another A DNA sequence between the Alu “5,” ends (“tail-to-head” orientation).
  • Figure 7 is a cross-Alu PCR amplification of a single oligonucleotide primer located at the tail of AluY observed in an ultraviolet lamp, in Example 2, tumor tissue DNA replicon and control peripheral blood DNA replicon on agarose gel
  • Two samples labeled F were images of tumor tissue DNA and peripheral blood DNA amplification products in a primary glioblastoma patient in an agarose gel.
  • Two samples labeled L were images of tumor tissue DNA and peripheral blood DNA amplification products in an agarose gel from another primary glioblastoma patient.
  • Two samples labeled G were images of tumor tissue DNA and peripheral blood DNA amplification products in agarose gel in patients with secondary glioblastoma.
  • Two samples labeled W were images of tumor tissue DNA and peripheral blood DNA amplification products in agarose gel in patients with dysplastic gliomas.
  • the final band is the DNA molecular marker (lkb) (produced by Fermentas).
  • the arrow indicates that the replication error phenotype appears in each subtype of glioblastoma.
  • Figure 8 is a cross-sectional oligodendromas tumor tissue DNA replicon and control peripheral blood DNA after three-Alu mixed oligonucleotide primers inter-Alu PCR amplification observed in an ultraviolet lamp in Example 2.
  • An image of the replicon in an agarose gel (both white patchy shadows).
  • On the left is the image of the DNA amplification replicon of the tumor tissue in the agar gel, and the middle is the image of the control peripheral blood DNA amplification replicon in the agar gel; the right side is the DNA molecular marker (lkb) (produced by Fermentas) .
  • Figure 9 is a schematic diagram showing the position of the primers in the AluY structure and the direction of amplification in the AluY common sequence, in which two sequences containing no CpG sites in the AluY common sequence are selected.
  • One of the oligonucleotide primers was named CH11, and its amplification direction was toward “5'” and the length was l lbp.
  • the oligonucleotide primer can be amplified in a "head-to-head” orientation to obtain a DNA sequence between the Alu "5'” end and the other Alu "5'” end.
  • Name another oligonucleotide primer as CT11 with the direction of amplification toward "3'”. The length is l lbp.
  • the oligonucleotide primer can be amplified in a "tail-to-tail” direction to obtain a DNA sequence between the Alu "V” end and the other Alu “3'” end.
  • Two oligonucleotide primers, CH11 and CT11 act on the same inter-Alu PCR reaction to amplify a DNA sequence between the Alu "5" end and another Alu “3'” end ("head to tail” Direction), and the DNA sequence between the Alu "3 '" end and the other Alu "5 '" end ("tail-to-head” direction).
  • Figure 10 shows the specific position and sequence of "C” "T” in the sequence of two AluY common sequence oligonucleotide primers in Example 3. After the two AluY common sequence oligonucleotide primers were converted to "T", their sequences were: CH1 "5' TTTAATAAAAA 3' ", CT11 "5' AACATCAAAAT 3' "o
  • Figure 11 shows the directionality of the amplified genomic DNA of two Alu oligonucleotide primers which were converted to "T” by “C” in Example 3. It is identical to the inter-Alu PCR amplification direction of the three oligonucleotide primers shown in Figure 6.
  • two different directions of AluY common sequence oligonucleotide primers amplify genomic DNA of bisulfite pretreated tumor tissue and control tissue (including peripheral blood), which can produce the above four different Amplification product.
  • Figure 12 is a graph showing the results of DNA sequencing after pretreatment with bisulfite in Example 3.
  • High-throughput sequencing instruments based on next-generation sequencing technology require only 3 g of PCR product to detect DNA sequences pretreated with bisulfite.
  • the methylated “C” does not change, not The basic "C” will be transformed into! 1 .
  • the SNPs detected are SNPs that are concentrated in the gene region in haploid or homozygous diploid or heterozygous diploid human genomic DNA.
  • Figure 1 shows an example of such a SNP in the gene region. In order to obtain the sequence of the gene region, it is necessary to concentrate the amplified gene region by inter-Alu PCR reaction, and then detect the SNP in the amplified gene region by a new generation sequencing technology.
  • Figure 2 shows the position of two oligonucleotide primers designed by the inter-Alu PCR of the present invention in a small fragment of the AluY consensus sequence in the Alu structure, and the two references. The direction of amplification of the object in genomic DNA; and Figure 3 shows the specific location and sequence of the two primers.
  • a single oligonucleotide primer can complement the detected template DNA at an appropriate sequence position by the action of a thermostable DNA polymerase to form a hybridized nucleic acid structure. Then, under this polymerization condition, the oligonucleotide primers are extended with four nucleotides (A, G, T, C), and the base-complementing principle is catalyzed by a thermostable DNA polymerase and amplified. The template chain is connected to achieve the purpose of copying the template chain. According to the directionality of Alu, one of the oligonucleotide primers can amplify Alu
  • the first step of the method is to extract human genomic DNA by phenol/chloroform method and purify it with ethanol.
  • Purified DNA was diluted to a certain concentration, according to the experimental requirements, DNA concentration is usually added 10 ⁇ 50 ⁇ ⁇ / ⁇ 1.
  • the selected oligonucleotide primers are located in the AluY consensus sequence. Among them, the oligonucleotide primers amplified in the "tail-to-tail" direction are located at positions 182 to 200 of the AluY common sequence, and the bases are
  • each PCR reaction system is 20 ⁇ 1, including 5 x Mastermix (10 x PCR amplification buffer (500 mM KC1, 100 mM) Tris-Cl, 15mM MgCl 2 ), 50mM MgCl 2 , and lOmM 4 kinds of dNTP mixture) 4 ⁇ 1, 5 ⁇ "tail-to-tail” or "head-to-head” direction amplification of oligonucleotide primers ⁇ , thermostable DNA polymerase ⁇ . ⁇ , 50ng/ul ⁇ 2 ⁇ 1 via human genome, and 12.9 ⁇ 1 deionized water.
  • the PCR amplification reaction consisted of: denaturation at 95 °C for 5 minutes, and then 35 amplification cycles including denaturation at 95 °C for 30 seconds.
  • the "head-to-head” amplification direction of the oligonucleotide primer required annealing at 66.3 °C for 30 seconds ("
  • the tail-to-tail "oligomer primer in the amplification direction requires annealing at 66.8 ° C for 30 seconds", extending at 72 ° C for 2 minutes; and finally ending the reaction at 72 ° C for 5 minutes.
  • Figure 4 is an image of the amplified DNA replicon in an agarose gel observed by an ultraviolet lamp.
  • the stepwise image produced by amplification is compared to the DNA molecular marker (lkb), showing that the amplified band is mainly between 300 bp and 2 kb.
  • the inter-Alu PCR amplification products in the above examples include 374 DNA fragments, of which 153 sequences are in the gene region, accounting for 40% of the DNA sequence obtained by sequencing; and the entire gene region is in the entire genome. The ratio was only 25%.
  • Alu is mainly concentrated in the gene region, and the sequence between the two Alus amplified by inter-Alu PCR is also concentrated in the gene region.
  • the number of genes in the entire genome is about 25,000, and the number of tumor-related genes is currently known to be 6522, accounting for 26% of the total number of genes.
  • the gene sequences in this example are located in 128 genes, of which 75 genes are tumor-associated genes, accounting for 58% of the total number of genes covered by sequencing. Comparing human genome sequences with BLAST and UCSC databases, the number of SNPs including non-gene regions was 262, of which 42 were new SNPs. Therefore, it is proved that this method can provide more information on the SNP of the gene region.
  • Example 2 is similar to Example 1, except that this example focuses on the study of the relevance of polygenic diseases, using this method to detect the relationship between SNPs, insertions/deletions and diseases in the gene region, especially through “exons”. Capture “analyzes the relationship between SNPs, insertions/deletions and disease in exons of the gene region.
  • the sequence near the "3'" end of the AluY common sequence was selected as the oligonucleotide primer, and the amplification direction of the primer was selected as the oligonucleotide primer, and the amplification direction of the primer was
  • Figure 5 shows the sequence of the oligonucleotide primer and its position in AluY. The positions of the oligonucleotide primers range from position 278 to position 295 in the Alu consensus sequence. Base is
  • any oligo can be complementary to the template DNA being detected at appropriate sequence positions to form a hybrid nucleic acid structure.
  • oligonucleotide primers are extended with four nucleotides (A, G, T, C), and the base-complementing principle is catalyzed by a thermostable DNA polymerase and amplified.
  • the template chain is connected to achieve the purpose of copying the template chain.
  • Figure 6 shows the amplification direction of the three oligonucleotide primers. According to the directionality of Alu, an oligonucleotide primer of AluY common sequence and Alu common sequence oligonucleotide primer R12A/267 can amplify between one Alu "3 '" end and another Alu "3 '" end.
  • oligonucleotide primer can amplify a DNA sequence between the Alu "5'” end and another Alu “5'” end (" Head to head “direction”.
  • Mixed oligonucleotide primers can also amplify a DNA sequence between the Alu "5'” end and another Alu "3'” end ("head-to-tail” orientation), and an Alu "3'” end with another A DNA sequence between the ends of the Alu "5'"("tail-to-head” orientation).
  • Single-AluY "tail-to-tail" amplified oligonucleotide primers can amplify tumor tissue DNA and control tissue DNA by inter-Alu PCR, and the difference between the two sets of amplification products is shown by DNA agarose gel electrophoresis. That is, replication error ph. o
  • DNA replicons amplified by inter-Alu PCR of multi-oligonucleotide primers are detected by high-throughput sequencing instruments, and tumor-related gene SNPs, point mutations, insertions/ Missing fragment information.
  • the first step is to extract DNA from patient tumor tissues and tumor control tissues (including peripheral blood) by phenol/chloroform method and purify with ethanol.
  • the purified DNA was diluted to a certain concentration, and the DNA concentration was 5 ⁇ !3 ⁇ 4/ ⁇ 1 as required by the experiment.
  • the selected oligonucleotide primers are located at the tail of the AluY common sequence, and their positions and base sequences are as described above, and are positions 278 to 295 in the AluY common sequence.
  • the base sequence is "5'-GAGCGAGACTCCGTCTCA-3'", and the "tail-to-tail" direction is amplified.
  • the total volume of each PCR reaction system is 20 ⁇ l, including 5 x Mastermix (10 x PCR amplification buffer (500 mM KC1, 100 mM Tris-Cl, 15 mM MgCl 2 ), 50 mM MgCl 2 , and 10 mM 4 kinds of dNTP mixture) 4 ⁇ 1, 5 ⁇ Primer AluYTl 1.2 ⁇ 1, thermostable DNA polymerase 0.1 ⁇ 1, 50 ⁇ / ⁇ 1 via human genome ⁇ 2 ⁇ 1, and deionized water 12.7 ⁇ 1.
  • 5 x Mastermix 10 x PCR amplification buffer (500 mM KC1, 100 mM Tris-Cl, 15 mM MgCl 2 ), 50 mM MgCl 2 , and 10 mM 4 kinds of dNTP mixture
  • thermostable DNA polymerase 0.1 ⁇ 1 50 ⁇ / ⁇ 1 via human genome ⁇ 2 ⁇ 1, and deionized water 12.7 ⁇ 1.
  • the PCR amplification reaction included: denaturation at 95 ° C for 5 minutes, and then 35 amplification cycles including denaturation at 95 ° C for 30 seconds, oligonucleotide primers required annealing at 67 ° C for 30 seconds, 72 ° C extension for 2 minutes; The reaction was terminated by extension of °C for 5 minutes.
  • 2 ( ⁇ L was subjected to 1.5% agarose gel electrophoresis, and the form and quality of the amplified product were observed under ultraviolet light by staining with ethidium bromide.
  • Fig. 7 shows the expanded tumor observed by ultraviolet lamp. Image of tissue DNA replicon and control peripheral blood DNA replicon in agarose gel, arrow indicates that replication error phenotype appears In each subtype of glioblastoma.
  • SNPs tumor-related gene region
  • point mutations point mutations
  • insertion/deletion fragment information inter-Alu PCR reactions involving multiple Alu family common sequence oligonucleotide primers
  • centralized amplification of gene regions in the genome and DNA replication
  • DNA replication The relationship between SNPs, point mutations, insertions/deletions and tumors was detected by high-throughput sequencing instruments based on next-generation sequencing technology. Specifically, the above three oligonucleotide primers are mixed in 5:3:1 and added to the PCR reaction.
  • each PCR reaction system is 20 ⁇ l, including 5 x Mastermix (10 x PCR amplification buffer (500 mM KC1, 100 mM Tris-Cl, 15 mM MgCl 2 ), 50 mM MgCl 2 , and lOmM 4 and dNTP mixture) 4 ⁇ 1, 5 ⁇ AluY common sequence "tail-to-tail” amplification direction oligonucleotide primer 1.5 ⁇ 1, 5 ⁇ AluY common sequence "head-to-head” amplification direction oligonucleotide primer 0.9 ⁇ 1, 5 ⁇ ⁇ 1 ⁇ common sequence "tail-to-tail” amplification direction oligonucleotide primer R12A/267 0.3 ⁇ 1, thermostable DNA polymerase ⁇ . ⁇ , lOng/ ⁇ human genomic DNA 1 ⁇ 1, and deionized water 12.2 ⁇ 1.
  • 5 x Mastermix 10 x PCR amplification buffer (500 mM KC1, 100 mM Tris-Cl, 15 mM M
  • the PCR amplification reaction included: denaturation at 95 ° C for 5 minutes, and then 20 amplification cycles including denaturation at 95 ° C for 30 seconds, multiple oligonucleotide primers required annealing at 57.8 ° C for 30 seconds, 72 ° C extension for 2 minutes; The reaction was terminated by extension of °C for 5 minutes. After the reaction was completed, 5 L and 50% glycerol were mixed and subjected to 1.5% agarose gel electrophoresis, and the morphology and quality of the amplified product were observed under an ultraviolet lamp.
  • Figure 8 is an image of amplified tumor tissue DNA replicon and control peripheral blood DNA replicon in an agarose gel observed by an ultraviolet lamp, which is a white patchy shadow.
  • the DNA replicon satisfies the detection requirements of high-throughput sequencing instruments, generating more sequence-specific sequences than in Example 1, for analyzing SNPs, point mutations, insertion/deletion and tumor relationships in the tumor genome region.
  • the assembly of the small fragment oligonucleotide analysis software package can also be used to form long-chain DNA sequences, and the positions of the detected DNA sequences can be determined by comparing human genome sequences.
  • each sequence is compared to the human genome sequence by BLAST and UCSC databases, and the difference of SNPs in each sequence between the tumor and the control genomic DNA, the location of the point mutation, and the location of the DNA insertion/deletion of the tumor tissue can also be determined.
  • the poly-nucleotide primer inter-Alu PCR product is purified, and the "exon capture” technique is used to analyze only SNPs, point mutations, insertions/deletions and tumors in the exon of the gene region.
  • the vector pETV-SD which captures the exon is a retroviral shuttle vector (shuttle vec t 0 r). Because all genes with introns and exons are passed after transcription.
  • RNA splicing which requires a splicing donor (SDM apex and splicing acceptor (SA) site. After the DNA replicon is interrupted by ultrasound into small fragments, the cloning is in the vector at the exon. The capture sequence is "downstream of the cloning site.”
  • SA splicing donor
  • the ⁇ 2 cell provides a protein product such that the vector (which cannot itself synthesize a viral protein) becomes a retrovirus that proliferates in the cell.
  • the retrovirus When the retrovirus is transcribed intracellularly, if the insert contains a functional SA site, an RNA splicing reaction may occur and the IVS is excised. Both spliced and unspliced viral RNA are packaged in a virion, collected from cell culture fluid and used to infect the ampho tropic retroviral packaging cell line PA-317. This allows the retrovirus to undergo another round of replication and produce a high-cost virus stock that can infect the monkey kidney cell line COS cells.
  • the virus isolated from the second cell line PA-317 was used to infect COS cells constitutively producing SV40T (tumor) antigen.
  • the viral RNA genome is reverse transcribed and replicated as a circular DNA episome under the action of the SV40 origin of replication on the vector.
  • the replicated episomal DNA was recovered from COS cells and digested with restriction endonuclease Dpnl to transform the bacteria. Transformants were selected on a medium containing kanamycin (Kn) and 5-chloro-4-bromo-3-mercapto- ⁇ -D-galactoside (X-gal). Lu galactosidase hydrolyzes X-gal to form a blue product.
  • each sequence is compared to the human genome sequence by BLAST and UCSC databases, and the difference of SNPs in each sequence between the tumor and the control genomic DNA, the location of the point mutation, and the location of the DNA insertion/deletion of the tumor tissue can also be determined.
  • Embodiment 2 This embodiment is similar to Embodiment 1 and Embodiment 2. Because the vast majority of 5-methylcytosine (5mC) is mainly concentrated in the Alu family of repeats rich in CpG sites, and it is estimated that the Alu family can contain up to 33% of CpG sites throughout the human genome. . Previous studies have shown that in tumors In the genome, the methylation level of some specific CpG sites in the Alu sequence is significantly reduced, which is particularly prominent in the AluY subfamily. In addition, in studies related to schizophrenia, we found that the level of methylation of certain CpG sites in and around the AluY sequence also changed significantly.
  • 5mC 5-methylcytosine
  • the nucleotide primer cannot include the CpG site and the selected position is required.
  • the "C” in the non-CpG site in the conversion is converted to "T”.
  • the selected AluY common sequence needs to be closer to the intermediate region of the common sequence. As shown in Fig.
  • oligonucleotide primers Two sequences of the AluY common sequence near the intermediate region containing no CpG sites were selected as primers.
  • One of the oligonucleotide primers was named CH11, and its amplification direction was toward "5'” and the length was llbp.
  • the oligonucleotide primer can be amplified in a "head-to-head” orientation, ie, a DNA sequence between one Alu "5,” end and another Alu "5,” end.
  • CT11 Another oligonucleotide primer was named CT11 with a direction of amplification toward "3'" and a length of llbp.
  • the oligonucleotide primer can be amplified in a "tail-to-tail” direction, ie, a DNA sequence between one Alu "3'” end and another Alu “3'” end.
  • Two oligonucleotide primers, CH11 and CT11 act on the same inter-Alu PCR reaction to amplify a DNA sequence between the Alu "5'” end and the other Alu”3'" end ("head to tail") Direction), and the DNA sequence between the Alu "3'" end and the other Alu "5,” end (“tail-to-head” direction).
  • the amplification products include DNA replicons in four directions: head-to-head, tail-to-tail, head-to-tail, and tail-to-head.
  • the amplified product was visualized as a white patch by DNA agarose gel electrophoresis. Specifically, 900 ng of genomic DNA was added to 0.3 M sodium hydroxide (NaOH) and placed at 42 ° C for 20 minutes. It was then placed at 95 °C for 3 minutes and at 0 °C for 1 minute. Thereafter, the above sample was added with sodium metabisulfite (2.0 M) and hydroquinone (0.5 mM) having a pH of 5.0, and the upper layer was covered with mineral oil and placed at 55 ° C for 16 hours.
  • the bisulfite pretreated DNA is purified and amplified by an inter-Alu PCR reaction. The total volume of each PCR reaction system is 20 ⁇ 1, including 5 x Mastermix (10 x PCR amplification buffer)
  • the PCR amplification reaction includes: denaturation at 95 °C for 5 minutes, and the subsequent 20 amplification cycles include denaturation at 95 °C for 30 seconds, annealing at 52 °C for 30 seconds, extension at 72 °C for 2 minutes, and termination at 72 °C for 5 minutes. reaction. Since the bisulfite-pretreated genomic DNA is single-stranded DNA, it is not easily amplified in the inter-Alu PCR reaction. Therefore, the above inter-Alu PCR reaction needs to be repeated to increase the quality of the DNA replicon.
  • the assembly of (SOAPalinger) forms long-chain DNA sequences and determines the location of each detected DNA sequence by comparing human genome sequences.
  • each sequence is aligned with the human genome sequence by BLAST and UCSC databases to determine the CpG sites in the gene region tumors and control genomic DNA in each sequence.
  • Example 3 can also be used for the exploration of changes in the methylation level of other diseases, that is, although in Example 1, the sequencing results of the high-throughput sequencing instrument show that 58% of the gene region sequences belong to the tumor Related genes, but all genes have the possibility of being associated with various diseases, so this method can be extended to study the characteristics of other disease gene regions.

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Description

以 Alu间聚合酶链式反应为基础的检测基因区特征的方法 技术领域
本发明属于生物技术领域。 具体地来说，是涉及集中检测基因区单核苷酸多 态性 （SNP)、 点突变， 序列插入 /缺失及双脱氧核苷酸分子 （DNA) 甲基化 CpG 位点的方法。 该方法以 Alu家族， 特别是 AluY亚家族共同序列为主要寡核苷酸 引物， 利用多个引物 Alu间聚合酶链式反应 （inter-Alu PCR)扩增基因组 DNA， 单次扩增 DNA 复制子多集中于基因组中各基因区。 该方法的特征在于， 通过 inter-Alu PCR可过滤掉部分非基因区序列，使新一代测序技术集中检测基因组中 基因区的 SNP、 点突变， 序列插入 /缺失及 DNA甲基化 CpG位点， 并可节省测 序所需基因组 DNA用量。
背景技术
以新一代测序技术循环阵列合成测序法为基础设计的高通量测序仪，可以在 单次测序中产生百万个碱基的序列。这一生物技术的进步， 极大程度上减少了全 基因组测序的费用， 并且大大加快了疾病病因学研究的速度。该技术现用于包括 肿瘤， 精神疾病在内的多种疾病的相关性研究中。然而在单次测序中， 需要至少 3μ§基因组 DNA, 并且 3μ§通常不能满足全基因组测序的要求。高 DNA总量的 需求会降低样本提供者的认同度。此外， 由于高通量测序仪随机测序的特点， 单 次测序所得的数据可能只集中于基因组中的某些区域，而丢失掉研究所需要的其 它重要区域。 依据人类基因组计划所提供的数据， 基因组中只有 1 %的区域为基 因编码区， 而整个基因区只占基因组的 25 %。 也就是说， 高通量测序仪测序所 产生的数据中，只有少数涵盖对于疾病病因学研究比较重要的基因区序列。因此， 当前的方法仍存在成本一质量一数量不均衡的问题。
新方法需减少测序所需 DNA用量，增加数据有效性， 同时进一步降低成本。 聚合酶链式反应（PCR)因其可呈指数级扩增模板 DNA，所以可以降低模板 DNA 用量。 然而， 单一基因区扩增所使用的 PCR技术会局限所获得信息的数量， 多 对引物的多次 PCR反应也会造成工作量成倍增加。 美国专利 5， 773， 649、 6， 060， 243及 7， 537， 889均涉及到可以利用 Alu间聚合酶链式反应（inter-Alu PCR) 扩增人类基因组 DNA中多个区域。其中，专利 5， 773， 649是利用 inter-Alu PCR 技术扩增肿瘤基因组 DNA及同一患者外周血 DNA， 并通过 DNA琼脂糖凝胶电 泳观察肿瘤基因组 DNA复制错误 （replication error) 表型。 但是， 具体在该疾 病中基因组中哪些区域发生了突变并没有进行深入研究。
研究表明， 多种遗传疾病与 AluY亚家族插入基因组中造成基因组不稳定有 关； 同时， 该亚家族作为重组热点区， 其结构内部及周围区域的 SNP可能为疾 病相关 SNP。 另夕卜， 在整个人类基因组， Alu家族可包含多达 33 %的 CpG位点。 且既往研究表明， 在肿瘤基因组中， Alu序列中的某些特殊 CpG位点的甲基化 水平出现显著性降低， 这一现象在 AluY亚家族中尤为突出。 因此， 以 AluY共 同序列为寡核苷酸引物的 inter-Alu PCR, 可以实现集中于基因区 SNP、 点突变， 插入 /缺失及 DNA CpG位点甲基化水平的检测。这一改进的方法可以满足在疾病 病因学研究中使用微量 DNA就可以通过高通量测序仪器获得更多有效的测序数 据， 满足研究对成本一质量一数量均衡的要求。
发明内容
本发明涉及集中检测基因区单核苷酸多态性 （SNP)、 点突变， 插入 /缺失及 双脱氧核苷酸分子（DNA) 甲基化 CpG位点的方法。 总体上说， 该方法是通过 inter-Alu PCR, 使新一代测序技术可集中检测基因组中基因区序列特征的方法。
依据 Alu重复序列集中于基因区这一特点， 通过多引物 inter-Alu PCR扩增 基因组中多个两 Alu序列间区域，所得纯化 PCR产物的质量为模板基因组 DNA 质量的 6倍； 并且， 该 PCR产物为涵盖基因组中多个基因区域的 DNA复制子。 此方法可保证新一代测序技术仅使用纳克级基因组 DNA， 便可以集中检测基因 区的 SNP、 点突变， 序列插入 /缺失及 DNA甲基化 CpG位点。
在本发明的一个实施方案中， 根据 AluY亚家族的特征， 即多种遗传疾病经 研究与 AluY亚家族插入基因组中造成基因组不稳定有关， 另外， 该亚家族作为 重组热点区， 其结构内部及周围区域的 SNP可能为疾病相关 SNP; 因而寡核苷 酸引物设计依据上述 AluY的特征， 选取以 AluY亚家族共同序列中的片段作为 寡核苷酸引物。在 Alu间聚合酶链式反应（inter-Alu PCR)中， 通过热稳定 DNA 聚合酶的作用， AluY共同序列小片段寡核苷酸引物便可以与被检测的模板 DNA 在适当的序列位置互补， 形成杂交的核酸结构。 然后， 在此聚合条件下， 以四种 核苷酸 （A、 G、 T、 C) 延伸寡核苷酸引物， 以碱基互补原则， 在热稳定 DNA 聚合酶的催化下与需要扩增的模板链进行连接， 从而达到复制模板链的目的。根 据 PCR的扩增特点， 即 DNA的复制呈指数级扩增， 也就是说 PCR产物的质量 要多于原模板 DNA的质量。在本发明中，纯化后的 PCR产物质量为原模板质量 的 6倍。 同时， 因为 Alu重复序列的结构相似， 且在基因组中的数量超过 10%， 所以单一 AluY共同序列小片段寡核苷酸引物可通过 inter-Alu PCR扩增基因组中 多个区域， 得到多个大小不等的 DNA复制子。 经过 DNA琼脂糖凝胶电泳， 通 过溴乙锭染色， 在紫外灯下观察扩增产物形态为白色阶梯状影。 如 PCR反应中 的小片段寡核苷酸引物为多个， 则紫外灯下扩增产物形态为白色斑片状影。在本 发明中， 因为 Alu重复序列多出现在基因组中的基因区， 所以以 AluY共同序列 小片段寡核苷酸引物 inter-Alu PCR扩增的 DNA复制子在基因区的比率要占到 40%； 而整个基因区在整个基因组中的比率只有 25 %。 这样便可仅用 3μ§纳克 级的 DNA模板扩增的 inter-Alu PCR产物放入高通量测序仪器中进行测序，所得 的多位点 SNP主要集中于基因区的 Alu片段及 Alu周围片段中。 因为约 50%的 SNP分布于重复序列区，其余则分布于重复序列以外的非编码序列区和编码基因 区。因此，该方法可用于检测基因组中，特别是基因区的多个 SNP及发现新 SNP。
本发明的另一个实施方案是利用上述方法检测不同疾病，特别是癌症基因组 中基因区内含子及外显子的点突变， 插入 /缺失。 在整个基因组中基因数量约为 25， 000个， 而现已知肿瘤相关基因数量为 6522个， 占整个基因总数的 26%。 在本发明中， 通过以 AluY共同序列小片段为寡核苷酸引物的 inter-Alu PCR, 扩 增肿瘤基因组 DNA， 所得的基因区序列所在的基因有 58 %为肿瘤相关基因。 因 此， 可采用包括不同扩增方向的 AluY共同序列为寡核苷酸引物及 "尾对尾"扩增 方向的 Alu共同序列寡核苷酸引物（R12A/267)作为混合引物，在 inter-Alu PCR 中， 通过热稳定 DNA聚合酶的作用， 多寡核苷酸引物与被检测的模板 DNA在 适当的序列位置互补， 形成杂交的核酸结构。 然后， 在此聚合条件下， 以四种核 苷酸 （A、 G、 T、 C) 延伸寡核苷酸引物， 以碱基互补原则， 在热稳定 DNA聚 合酶的催化下与需要扩增的模板链进行连接， 从而达到复制模板链的目的。 因为 多个引物共同作用于同一 PCR反应体系中， 所以所得 DNA产物经过 DNA琼脂 糖凝胶电泳， 通过溴乙锭染色， 在紫外灯观察下扩增产物形态为白色斑片状影。 按照如前所述技术，可检测 inter-Alu PCR扩增区域中包含的 SNP是否为肿瘤相 关性 SNP， 同时可将 DNA复制子做进一步打断处理， 结合外显子捕获技术， 着 重分析扩增区域外显子中的 SNP与肿瘤的相关性。
本发明的另一个实施方案是利用上述方法进行基因区 DNA甲基化水平的检 测。 DNA中的胞嘧啶 （C) 甲基化修饰多发生于 5'-CpG-3' "C"±， 生成 5-甲 基胞嘧啶 (5mC)。 绝大多数的 5mC主要集中于富含 CpG位点的重复序列 Alu家 族中， 并且据估计， 在整个人类基因组， Alu家族可包含多达 33 %的 CpG位点。 所以， 以 AluY共同序列中不含 CpG位点的序列设计 2个不同扩增方向的寡核 苷酸引物， 将引物中的" C"全部转换成" T"。 通过 inter-Alu PCR扩增经亚硫酸氢 盐预处理的肿瘤及正常对照组织 （外周血） 基因组 DNA， 可获得基因组中富含 CpG位点的 Alu及其周围区域的 PCR产物。 2个不同扩增方向的寡核苷酸引物 共同作用于同一 inter-Alu PCR扩增反应体系， 可获得包括了 "尾对尾"、 "头对 头"、 "头对尾"及"尾对头 "4个方向的 DNA复制子，进一步增加了涵盖 CpG位点 区域的数量，可使经新一代测序技术测序所获得的序列尽量多地提供肿瘤基因组 CpG位点甲基化水平改变程度的信息。
在不背离本发明公开精神的前提下，对本发明所做的各种替换和修饰， 都将 落入本发明范围内。
本说明书中使用的术语"基因区" 是指基因组序列中基因所在的区域。 基因 (遗传因子） 是遗传的物质基础， 是 DNA分子上具有遗传信息的特定核苷酸序 列的总称， 是具有遗传效应的 DNA分子片段。
本说明书中使用的术语"纯化后 PCR产物" 是指在经过 inter-Alu PCR反应 后， 将 PCR产物通过乙醇或纯化试剂盒去掉多余的引物、 酶、 矿物油、 甘油， 盐等杂质后所得的纯化物。
本说明书中使用的术语 "两个 Alu序列间的区域" 是指在 inter-Alu PCR扩增 产物所位于的区域。 由于 Alu序列在基因组中分布较为广泛， 在热稳定 DNA聚 合酶可以扩增的 DNA长度容许下，扩增所包括的相邻两个 Alu序列之间的 DNA 序列。
本说明书中使用的术语"质量" 是含有两个方面的意思。第一，指在 inter-Alu PCR扩增产物的多少。 第二， 指测序数据满足研究要求的程度。
本说明书中使用的术语 "DNA复制子 "是指 inter-Alu PCR扩增产物， 这里是 以 Alu寡核苷酸引物扩增基因组 DNA所得的 PCR产物。
本说明书中使用的术语 "新一代测序技术"是指比第一代测序技术（荧光标记 的 Sanger法） 更为先进的循环阵列合成测序法。 该技术可以在单次测序中获得 数百万碱基的序列。
本说明书中使用的术语"纳克级基因组 DNA"是指实验中常用， 汉语翻译出 的 DNA质量的单位。 其英语拼写为 "ng"。
本说明书中使用的术语 "AluY共同序列小片段寡核苷酸引物"是指选取 AluY 亚家族共同序列中的 10— 20个碱基作为引物， 并应用于 inter-Alu PCR反应中。 这些引物的片段可以按照不同实验的要求，从 AluY共同序列中的任意位置选取。
本说明书中使用的术语"白色阶梯状影 "是指单个或多个寡核苷酸引物的 inter-Alu PCR反应所产生的 DNA复制子， 经过 DNA琼脂糖凝胶电泳， 通过溴 乙锭染色， 在紫外灯下所显示的形态。 其颜色为白色， 形状如阶梯。
本说明书中使用的术语"白色斑片状影" 是指多个寡核苷酸引物的 inter-Alu PCR反应所产生的 DNA复制子， 经过 DNA琼脂糖凝胶电泳， 通过溴乙锭染色， 在紫外灯下所显示的形态。 其颜色为白色， 形状云翳或斑片。
本说明书中使用的术语"热稳定 DNA聚合酶 "是指用于 inter-PCR反应的 DNA聚合酶。 该热稳定 DNA聚合酶可以是 Taq酶， KOD酶及其他各种可以用 于各种 PCR反应的 DNA聚合酶。
本说明书中使用的术语"不同扩增方向 "是指按照 Alu具有 "5， "端头部及 "3 ' "端尾部， inter-Alu PCR的扩增方向可以是朝向" 5 ' "端，也可以朝向" 3 ' " 端。 因此 inter-Alu PCR扩增可以出现不同方向。
本说明书中使用的术语" '尾对尾 ' "是指按照 Alu具有" 5 ' "端头部及" 3 ' " 端尾部， inter-Alu PCR扩增一个 Alu " 3 ' "端尾部与一个 Alu " 3 ' "端尾部间 的 DNA区域。
本说明书中使用的术语" '头对头 ' "是指按照 Alu具有" 5 ' "端头部及" 3 ' " 端尾部， inter-Alu PCR扩增一个 Alu "5， "端头部与一个 Alu "5， "端头部 间的 DNA区域。
本说明书中使用的术语" '头对尾 ' "是指按照 Alu具有" 5 ' "端头部及" 3 ' " 端尾部， inter-Alu PCR扩增一个 Alu "5 ' "端头部与一个 Alu "3 ' "端尾部 间的 DNA区域。
本说明书中使用的术语" '尾对头 ' "是指按照 Alu具有" 5 ' "端头部及" 3 ' " 端尾部， inter-Alu PCR扩增一个 Alu "3 ' "端尾部与一个 Alu "5， "端头部 间的 DNA区域。
本说明书中使用的术语"外显子捕获"是指构建一种载体， 从其插入片段中 识别和回收外显子序列，从而克隆目的基因。在本发明中，主要将 inter-Alu PCR 产物作为外显子捕获的模板。
本说明书中使用的术语 "CpG位点"是指 DNA中的胞嘧啶 （C) 通过磷酸 二酯键与鸟嘌呤 （G) 形成线性连接。 在哺乳动物中 CpG位点中的 "C" 甲基化 水平为 70%到 80%。
本发明涉及集中检测基因区特征的方法。该方法是通过 inter-Alu PCR,使测 序集中于检测基因组中基因区序列的方法。 依据以 AluY共同序列为寡核苷酸引 物的 inter-Alu PCR扩增基因组中多个两 Alu序列间区域， 可获得高质量的纯化 PCR产物； 并且， 该 PCR产物为涵盖基因组中多个基因区域的 DNA复制子。 此方法可保证新一代测序技术仅使用纳克级基因组 DNA， 达到集中检测基因区 特征的目的。
在本发明的一个实施方案中， 根据 AluY亚家族的特征， 即 AluY亚家族插 入基因组中造成基因组不稳定， 另外， 该亚家族作为重组热点区， 其结构内部及 周围区域的 SNP 可能为疾病相关 SNP; 因而寡核苷酸引物的设计需依据上述 AluY 的特征， 选取以 AluY 亚家族共同序列中的片段作为寡核苷酸引物。 在 inter-Alu PCR中， 通过热稳定 DNA聚合酶的作用， AluY共同序列小片段寡核 苷酸引物便可以与被检测的模板 DNA在适当的序列位置互补， 形成杂交的核酸 结构。 然后， 在此聚合条件下， 以四种核苷酸 （A、 G、 T、 C) 延伸寡核苷酸引 物， 以碱基互补原则， 在热稳定 DNA聚合酶的催化下与需要扩增的模板链进行 连接， 从而达到复制模板链的目的。根据 PCR的扩增特点， 即 DNA的复制呈指 数级扩增， 也就是说 PCR产物的质量要多于原模板 DNA的质量。 在本发明中， 纯化后的 PCR产物质量为原模板质量的 6倍。 同时， 因为 Alu重复序列的结构 相似， 且在基因组中的数量超过 10%， 所以单一 AluY共同序列小片段寡核苷酸 引物可通过 inter-Alu PCR扩增基因组中多个区域，得到多个大小不同的 DNA复 制子。 经过 DNA琼脂糖凝胶电泳， 通过溴乙锭染色， 在紫外灯观察扩增产物形 态为白色阶梯状影。 如 PCR反应中的小片段寡核苷酸引物为多个， 则紫外灯下 扩增产物形态为白色斑片状影。在本发明中， 因为 Alu重复序列多出现在基因组 中的基因区， 所以以 AluY共同序列小片段寡核苷酸引物 inter-Alu PCR扩增的 DNA复制子在基因区的比率要占到 40%; 而整个基因区在整个基因组中的比率 只有 25 %。这样的扩增结果， 可以仅用 3纳克级 DNA， 便可以进行高通量测序， 所检测到的 SNP主要集中于基因区中。因为 SNP集中分布于重复序列区， 因此， 该方法可用于检测基因组中， 特别是基因区的多个 SNP及发现新 SNP。
本发明的另一个实施方案是利用上述方法检测不同疾病，特别是肿瘤基因组 中基因区内含子及外显子的点突变， 插入 /缺失。 在整个基因组中基因数量约为 25， 000个， 而现已知肿瘤相关基因数量为 6522个， 占整个基因总数的 26%。 在本发明中， 通过以 AluY共同序列小片段为寡核苷酸引物的 inter-Alu PCR, 扩 增肿瘤基因组 DNA， 所得的基因区序列所在的基因有 58 %为肿瘤相关基因。 因 此， 可采用包括不同扩增方向的 AluY共同序列或 Alu共同序列作为寡核苷酸引 物， 在 inter-Alu PCR中， 利用多个寡核苷酸引物混合物， 通过热稳定 DNA聚合 酶的作用， 多寡核苷酸引物与被检测的模板 DNA在适当的序列位置互补， 形成 杂交的核酸结构。 然后， 在此聚合条件下， 以四种核苷酸 （A、 G、 T、 C) 延伸 寡核苷酸引物， 以碱基互补原则， 在热稳定 DNA聚合酶的催化下与需要扩增的 模板链进行连接， 从而达到复制模板链的目的。 所得 DNA产物经过 DNA琼脂 糖凝胶电泳， 通过溴乙锭染色， 在紫外灯观察扩增产物形态为白色斑片状影。 按 照如前所述技术， 可检测 inter-Alu PCR扩增区域中与疾病相关的 SNP， 插入 / 缺失。 同时可将 DNA复制子做进一步打断处理， 结合外显子捕获技术， 着重分 析扩增区域外显子中的 SNP与肿瘤的相关性。
本发明的另一个实施方案是利用上述方法进行基因区 DNA甲基化水平的检 测。 利用重复序列 Alu家族中富含 CpG位点的特点， 以 AluY共同序列中不含 CpG位点的序列设计不同扩增方向的 2个扩增寡核苷酸引物， 将引物中的" C"全 部转换成" T"。 通过 inter-Alu PCR扩增经亚硫酸氢盐预处理的基因组 DNA， 可 获得基因组中多位点， 富含 CpG的 Alu及其周围区域的 PCR产物。 2个不同方 向的扩增引物共同作用于同一 inter-Alu PCR扩增反应体系， 可获得包括了 "尾对 尾"、 "头对头"、 "头对尾"及"尾对头 "4个方向的 DNA复制子， 可使经新一代测 序技术测序所获得的序列尽量多地提供基因组 CpG位点甲基化程度的信息。 这 一方法可用于正常人基因组甲基化水平的检测， 或对各种不同疾病状态下 DNA 甲基化水平改变程度进行分析。
以下， 结合附图和下列实施例对本发明作进一步描述。
附图说明
图 1是实施例 1中在基因区内 SNP的示意图。 显示本发明实施例 1中， 通 过 inter-Alu PCR反应， 使得寡核苷酸引物与基因组 DNA在适当位置结合后， 所 扩增出来的片段， 该片段主要位于基因区， 并且一个 SNP包含在该基因区中。
图 2是实施例 1中的 inter-Alu PCR以 AluY共同序列中的小片段设计的两个 寡核苷酸引物在 AluY结构中的位置， 及这两个引物在基因组 DNA中的扩增方 向的示意图。 因为 AluY具有 " 5 ' "端的头部， 及 " 3 ' "端的尾部， 并且它们 的分布在基因组中并不均匀， 所以适当距离的两个相邻 Alu 之间可以通过 inter-Alu PCR反应扩增出来。 也就产生了如图 2所示的， 朝向 " 5 ' "方向扩增 及朝向 "3 ' "方向扩增。
图 3是实施例 1中两个 AluY共同序列寡核苷酸引物在 AluY中的具体位置 和序列的示意图。其中， "尾对尾"方向扩增的寡核苷酸引物（AluT-T)位于 AluY 共同序列的第 182位到第 200位，碱基为" 5， -AGGCTGAGGCAGGAGAATG-3， ： "头对头"方向扩增的寡核苷酸引物 （AluH-H) 位于 AluY共同序列的第 66位 至 IJ第 86位， 碱基为 "5， -TGGTCTCGATCTCCTGACCTC-3 ' ，，。
图 4是实施例 1经紫外灯所观察到的扩增后 DNA复制子在琼脂糖凝胶中的 影像 (反色显示)图。 左侧为 "尾对尾" AluY寡核苷酸引物 inter-Alu PCR扩增产 物在琼脂糖凝胶中的影像， 如阶梯状； 中间为 DNA分子标记 （lkb) (Fermentas 公司生产）； 右侧为 "头对头" AluY寡核苷酸引物 inter-Alu PCR扩增产物在琼 脂糖凝胶中的影像， 如阶梯状。 两种寡核苷酸引物的 inter-Alu PCR所扩增出的 条带主要在 300bp到 2kb之间。 箭头所指的是切胶测序的条带位置。
图 5是实施例 2中， 靠近 AluY结构示意图尾部的寡核苷酸引物 （AluYTl ) 的序列及所在 AluY中的位置的示意图。 寡核苷酸引物的位置在 AluY共同序列 中的第 278位到第 295位。 碱基为 "5'-GAGCGAGACTCCGTCTCA-3' "。 它可 以用来显示肿瘤基因组 DNA复制错误表型。
图 6是 3个寡核苷酸引物的扩增方向示意图。依据 Alu的方向性，其中 AluY 共同序列的一条寡核苷酸引物和 Alu共同序列寡核苷酸引物 R12A/267可以扩增 一个 Alu "3 ' "端与另一个 Alu "3 ' "端之间的 DNA序列 （"尾对尾"方向）， 而另一条 AluY共同序列的寡核苷酸引物可以扩增一个 Alu "5 ' "端与另一个 Alu "5 ' "端之间的 DNA序列 （"头对头"方向）。 混合寡核苷酸引物还可以扩 增一个 Alu "5 ' "端与另一个 Alu "3 ' "端之间的 DNA序列（"头对尾"方向）， 以及一个 Alu "3 ' "端与另一个 Alu "5， "端之间的 DNA序列 （"尾对头"方 向）。
图 7是实施例 2中， 经紫外灯所观察到的位于 AluY尾部的单一寡核苷酸引 物 inter-Alu PCR扩增后， 肿瘤组织 DNA复制子与对照外周血 DNA复制子在琼 脂糖凝胶中的影像（反色显示）图。标记为 F的 2个样本为一名原发胶质母细胞 瘤患者的肿瘤组织 DNA与外周血 DNA扩增产物在琼脂糖凝胶中的影像。 标记 为 L的 2个样本为另一名原发胶质母细胞瘤患者的肿瘤组织 DNA与外周血 DNA 扩增产物在琼脂糖凝胶中的影像。 标记为 G的 2个样本为继发胶质母细胞瘤患 者的肿瘤组织 DNA与外周血 DNA扩增产物在琼脂糖凝胶中的影像。 标记为 W 的 2个样本为间变少枝胶质细胞瘤患者的肿瘤组织 DNA与外周血 DNA扩增产 物在琼脂糖凝胶中的影像。最后的条带为 DNA分子标记（lkb) (Fermentas公司 生产）。 箭头指出， 复制错误表型出现在胶质细胞瘤各亚型中。
图 8是实施例 2中，经紫外灯所观察到的 3个 Alu混合寡核苷酸引物 inter-Alu PCR扩增后，间变少枝胶质细胞瘤肿瘤组织 DNA复制子与对照外周血 DNA复 制子在琼脂糖凝胶中的影像图 （均为白色斑片状影）。 左侧为肿瘤组织 DNA扩 增复制子在琼脂凝胶中的影像， 中间为对照外周血 DNA扩增复制子在琼脂凝 胶中的影像； 右侧为 DNA分子标记 （lkb) (Fermentas公司生产）。
图 9是实施例 3中， 选取 AluY共同序列中靠近中间区域的两段不含 CpG 位点的序列作为引物在 AluY结构中的位置及扩增方向的示意图。 命名其中一 条寡核苷酸引物为 CH11 , 其扩增方向朝向 " 5' "， 长度为 l lbp。 该寡核苷酸引 物可以进行"头对头 "方向扩增， 即得到一个 Alu "5 ' "端与另一个 Alu "5 ' " 端之间的 DNA序列。命名另一条寡核苷酸引物为 CT11 ,其扩增方向朝向" 3' "， 长度为 l lbp。 该寡核苷酸引物可以进行 "尾对尾 "方向扩增， 即得到一个 Alu " V "端与另一个 Alu "3 ' "端之间的 DNA序列。 CH11和 CT11两个寡核 苷酸引物作用于同一个 inter-Alu PCR反应， 可以扩增一个 Alu " 5， "端与另一 个 Alu "3 ' "端之间的 DNA序列 （"头对尾"方向）， 以及一个 Alu "3 ' "端 与另一个 Alu "5 ' "端之间的 DNA序列 （"尾对头"方向）。
图 10显示的是实施例 3中， 2个 AluY共同序列寡核苷酸引物的序列中" C" 变 "T" 的具体位置及序列。 2个 AluY共同序列寡核苷酸引物经过将 "C"转 换成 "T"后， 它们的序列分别是： CH1 "5' TTTAATAAAAA 3' "， CT11 "5' AACATCAAAAT 3' "o
图 11显示的是实施例 3中， 2个经过 "C"转换成 "T"后的 Alu寡核苷酸 引物扩增基因组 DNA的方向性。其与图 6显示的 3个寡核苷酸引物的 inter-Alu PCR扩增方向相同。 可包括一个 Alu "3 ' "端与另一个 Alu "3 ' "端之间的 DNA序列 （"尾对尾"方向）， 一个 Alu "5 ' "端与另一个 Alu " 5， "端之间 的 DNA序列 （"头对头"方向）； 一个 Alu "5 ' "端与另一个 Alu "3 ' "端之 间的 DNA序列 （"头对尾"方向） 以及一个 Alu "3 ' "端与另一个 Alu "5 ' " 端之间的 DNA序列 （"尾对头"方向）。 所示实施例中， 2个不同方向的 AluY 共同序列寡核苷酸引物扩增经亚硫酸氢盐预处理后的肿瘤组织和对照组织 （包 括外周血） 基因组 DNA， 可产生上述 4种不同的扩增产物。
图 12显示的是实施例 3中， 经亚硫酸氢盐预处理后的 DNA测序结果示意 图。 以新一代测序技术为基础的高通量测序仪器仅需要 3 gPCR产物， 就可以 检测到经亚硫酸氢盐预处理后的 DNA序列， 甲基化的 " C"不会产生变化， 而 非甲基化的 "C"会转变成！1。
具体实施例
实施例 1
所检测的 SNP为单倍体或纯合子二倍体或杂合子二倍体人类基因组 DNA中 集中于基因区的 SNP。 图 1显示了这样一个在基因区内 SNP的例子。 为获得基 因区序列， 需要通过 inter-Alu PCR反应集中扩增基因区， 而后以新一代测序技 术检测扩增后基因区中的 SNP。图 2显示的是本发明中的 inter-Alu PCR以 AluY 共同序列中的小片段设计的两个寡核苷酸引物在 Alu结构中的位置，及这两个引 物在基因组 DNA中的扩增方向；而图 3显示的是这两个引物的具体位置和序列。 在 inter-Alu PCR中， 通过热稳定 DNA聚合酶的作用， 单一寡核苷酸引物便可以 与被检测的模板 DNA在适当的序列位置互补， 形成杂交的核酸结构。 然后， 在 此聚合条件下， 以四种核苷酸 （A、 G、 T、 C) 延伸寡核苷酸引物， 以碱基互补 原则， 在热稳定 DNA聚合酶的催化下与需要扩增的模板链进行连接， 从而达到 复制模板链的目的。 依据 Alu 的方向性， 其中一条寡核苷酸引物可以扩增 Alu
"3 ' "端与 Alu "3 ' "端之间的 DNA序列 （"尾对尾"方向）， 而另一条寡核 苷酸引物可以扩增 Alu "5 ' "端与 Alu "5 ' "端之间的 DNA序列 （"头对头" 方向）。 这些 DNA复制子经过高通量测序仪器检测， 达到集中检测基因组中基 因区 SNP及发现基因组中基因区新 SNP的目的。 下面描述用本发明的方法可以 集中检测基因区 SNP的实例。
方法的第一步是用苯酚 /氯仿法提取人类基因组 DNA， 并用乙醇纯化。 纯化 后的 DNA被稀释到一定浓度，按实验要求，所加 DNA浓度通常为 10〜50η§/μ1。 所选取的寡核苷酸引物位于 AluY共同序列中。 其中， "尾对尾"方向扩增的寡 核苷酸引物位于 AluY 共同序列的第 182 位到第 200 位， 碱基为
" 5 ' -AGGCTGAGGCAGGAGAATG-3 ' ； "头对头"方向扩增的寡核苷酸引物 位 于 AluY 共 同 序 列 的 第 66 位 到 第 86 位 ， 碱 基 为
"5 ' -TGGTCTCGATCTCCTGACCTC-3 ' 。 在 inter-Alu PCR反应中， 一个具体 实例为， 每个 PCR反应体系的总体积为 20μ1， 包括 5 x Mastermix (10 x PCR扩 增缓冲液（500mM KC1， lOOmM Tris-Cl, 15mM MgCl2), 50mM MgCl2， 及 lOmM 4 种 dNTP混合液） 4μ1，5μΜ "尾对尾"或 "头对头"方向扩增的寡核苷酸引物 Ιμΐ，热稳定 DNA聚合酶 Ο. ΐμΐ, 50ng/ul经人类基因组 ϋΝΑ 2μ1，及去离子水 12.9μ1。
PCR扩增反应包括： 95 °C变性 5分钟， 而后 35个扩增循环又包括 95 °C变性 30 秒， "头对头"扩增方向的寡核苷酸引物需要 66.3 °C退火 30秒 （"尾对尾"扩 增方向的寡核苷酸引物需要 66.8°C退火 30秒）， 72°C延伸 2分钟； 最后 72°C 延 伸 5分钟终止反应。 反应完毕后， 取 ΙΟμΙ^进行 1.5 %琼脂糖凝胶电泳， 通过溴 乙锭染色，在紫外灯下观察扩增产物的形态及质量。 图 4为经紫外灯所观察到的 扩增后 DNA复制子在琼脂糖凝胶中的影像。扩增所产生的阶梯状影像对比 DNA 分子标记（lkb)， 显示扩增条带主要在 300bp到 2kb之间。选取两个寡核苷酸引 物分别扩增人类基因组 DNA,并通过切胶的方法分离出 DNA复制子中的 7个大 小在 450bp到 2kb之间的 DNA复制子， 最终获得每个条带的总质量 >10μ§。 这 些 DNA复制子通过以新一代测序技术为基础的高通量测序仪器测出 372Mb的 DNA序列数据。此后， 借助小片段寡核苷酸分析软件包（SOAPalinger)的组装， 形成长链 DNA序列， 并通过对比人类基因组序列确定检测出的各 DNA序列的 位置。 另外， 各序列通过 BLAST及 UCSC数据库比对人类基因组序列， 明确 各序列中的 SNP及发现新 SNP。
经过测序及生物信息学处理， 上述实例中 inter-Alu PCR扩增产物包括 DNA 片段 374条， 其中 153条序列在基因区， 占测序所得 DNA序列的 40%; 而整个 基因区在整个基因组中的比率只有 25 %， 这结果证明了 Alu主要集中于基因区， 而通过 inter-Alu PCR扩增的两个 Alu之间的序列也多集中于基因区。 此外， 在 整个基因组中基因数量约为 25， 000个， 而现已知肿瘤相关基因数量为 6522个， 占整个基因总数的 26%。 在本实例中的所有基因区序列所在的基因为 128个， 其中 75 个基因为肿瘤相关性基因， 占总测序所涵盖的基因数量的 58 %。 通过 BLAST及 UCSC数据库对比人类基因组序列，发现包括非基因区在内的 SNP数 量为 262个， 其中新 SNP为 42个。 因此证明该方法可以提供更多量基因区 SNP 的信息。
实施例 2
实施例 2与实施例 1相似， 区别在于， 此实施例主要集中于对多基因疾病相 关性的研究， 利用该方法检测基因区 SNP、 插入 /缺失与疾病的关系， 特别是通 过 "外显子捕获"对基因区外显子中的 SNP、 插入 /缺失与疾病的关系进行分析。 为获得肿瘤基因组复制错误 （replication error) 的表型特征及更多测序数据， 选 取将 AluY共同序列中靠近 "3' "端的序列作为寡核苷酸引物， 引物扩增方向为
"尾对尾"。 图 5显示的是该寡核苷酸引物的序列及所在 AluY中的位置。 寡核 苷酸引物的位置在 Alu 共同序列中的第 278 位到第 295 位。 碱基为
"5'-GAGCGAGACTCCGTCTCA-3' "。 将此寡核苷酸引物与前述 "头对头"方 向扩增的寡核苷酸引物， 碱基为 "5 ' -TGGTCTCGATCTCCTGACCTC-3 ' "及 Alu共同序列引物 R12A/267, 碱基为 "5'-AGCGAGACTCCG-3' "按照一定比例 进行混合。在 inter-Alu PCR中， 通过热稳定 DNA聚合酶的作用， 任何一个寡核 苷酸引物便可以与被检测的模板 DNA在适当的序列位置互补， 形成杂交的核酸 结构。 然后， 在此聚合条件下， 以四种核苷酸 （A、 G、 T、 C) 延伸寡核苷酸引 物， 以碱基互补原则， 在热稳定 DNA聚合酶的催化下与需要扩增的模板链进行 连接，从而达到复制模板链的目的。图 6显示的是 3个寡核苷酸引物的扩增方向。 依据 Alu的方向性， 其中 AluY共同序列的一条寡核苷酸引物和 Alu共同序列寡 核苷酸引物 R12A/267可以扩增一个 Alu "3 ' "端与另一个 Alu "3 ' "端之间 的 DNA序列 （"尾对尾"方向）， 而另一条 AluY共同序列的寡核苷酸引物可以 扩增一个 Alu "5 ' "端与另一个 Alu "5 ' "端之间的 DNA序列 （"头对头"方 向）。 混合寡核苷酸引物还可以扩增一个 Alu "5 ' "端与另一个 Alu "3 ' "端之 间的 DNA序列 （"头对尾"方向）， 以及一个 Alu "3 ' "端与另一个 Alu "5 ' " 端之间的 DNA序列 （"尾对头"方向）。 单一 AluY "尾对尾"方向扩增的寡核 苷酸引物可通过 inter-Alu PCR扩增肿瘤组织 DNA和对照组织 DNA， 通过 DNA 琼脂糖凝胶电泳显示出两组扩增产物中的差异， 即复制错误表型 （replication error ) o 多寡核苷酸引物的 inter-Alu PCR扩增出的这些 DNA复制子经过高通量 测序仪器检测， 可获得与肿瘤相关的基因 SNP、 点突变， 插入 /缺失片段的信息。
一个具体实例为， 第一步是用苯酚 /氯仿法提取患者肿瘤组织和肿瘤对照组 织（包括外周血） DNA， 并用乙醇纯化。 纯化后的 DNA被稀释到一定浓度， 按 实验要求所加 DNA浓度为 5θ!¾/μ1。所选取的寡核苷酸引物位于 AluY共同序列 的尾部， 其位置与碱基序列如前所述， 为 AluY共同序列中的第 278位到第 295 位。 碱基序列为 "5'-GAGCGAGACTCCGTCTCA-3' "， "尾对尾"方向扩增。 在 inter-Alu PCR反应中， 每个 PCR反应体系的总体积为 20μ1， 包括 5 x Mastermix (10 x PCR扩增缓冲液（500mM KC1， lOOmM Tris-Cl, 15mM MgCl2)， 50mM MgCl2， 及 10mM 4种 dNTP混合液） 4μ1， 5μΜ 引物 AluYTl 1.2μ1，热稳定 DNA聚合酶 0.1μ1，50η§/μ1经人类基因组 ϋΝΑ 2μ1， 及去离子水 12.7μ1。 PCR扩增反应包括： 95°C变性 5分钟， 而后 35个扩增循环又包括 95°C变性 30秒，寡核苷酸引物需要 67°C退火 30秒， 72°C延伸 2分钟； 最后 72°C 延伸 5分钟终止反应。 反应完毕 后， 2(^L进行 1.5 %琼脂糖凝胶电泳， 通过溴乙锭染色， 在紫外灯下观察扩增 产物的形态及质量。 图 7为经紫外灯所观察到的扩增后肿瘤组织 DNA复制子与 对照外周血 DNA复制子在琼脂糖凝胶中的影像， 箭头指出， 复制错误表型出现 在胶质细胞瘤各亚型中。
进一步获得丰富的肿瘤相关的基因区 SNP、 点突变， 插入 /缺失片段信息， 需要多 Alu家族共同序列寡核苷酸引物参与的 inter-Alu PCR反应， 集中扩增基 因组中基因区， 所得 DNA复制子通过以新一代测序技术为基础的高通量测序仪 器检测基因区 SNP、 点突变， 插入 /缺失与肿瘤之间的关系。 具体为， 上述三个 寡核苷酸引物， 按 5: 3： 1混合后加入 PCR反应中。 在 inter-Alu PCR反应中， 每个 PCR反应体系的总体积为 20μ1， 包括 5 x Mastermix (10 x PCR扩增缓冲液 (500mM KC1， lOOmM Tris-Cl, 15mM MgCl2)， 50mM MgCl2，及 lOmM 4禾中 dNTP 混合液） 4μ1， 5μΜ AluY共同序列 "尾对尾 "扩增方向寡核苷酸引物 1.5μ1， 5μΜ AluY共同序列 "头对头 "扩增方向寡核苷酸引物 0.9μ1， 5μΜ Α1ιι共同序列 "尾 对尾"扩增方向寡核苷酸引物 R12A/267 0.3μ1，热稳定 DNA聚合酶 Ο.ΐμΐ, lOng/μΙ 人类基因组 DNA 1μ1， 及去离子水 12.2μ1。 PCR扩增反应包括： 95°C变性 5分 钟， 而后 20个扩增循环又包括 95°C变性 30秒， 多寡核苷酸引物需要 57.8°C退火 30秒， 72°C延伸 2分钟； 最后 72 °C 延伸 5分钟终止反应。 反应完毕后， 取 5 L 与 50%甘油混合后进行 1.5 %琼脂糖凝胶电泳， 在紫外灯下观察扩增产物的形态 及质量。 图 8为经紫外灯所观察到的扩增后肿瘤组织 DNA复制子与对照外周血 DNA复制子在琼脂糖凝胶中的影像， 为白色斑片状影。该实例中， 仅需要 750ng 的模板 DNA通过 inter-Alu PCR反应及纯化步骤， 即可产生大于 3μ§的 DNA复 制子。 该 DNA复制子可满足高通量测序仪器的检测要求， 生成比实例 1更多的 集中于基因区的序列， 用于分析肿瘤基因组基因区的 SNP、 点突变， 插入 /缺失 与肿瘤的关系。 同样可借助小片段寡核苷酸分析软件包（SOAPalinger) 的组装， 形成长链 DNA序列， 并通过对比人类基因组序列确定检测出的各 DNA序列的 位置。 另外， 各序列通过 BLAST及 UCSC数据库比对人类基因组序列， 确定 各序列中的 SNP在肿瘤与对照基因组 DNA中的差别， 点突变的位置， 也可以确 定肿瘤组织 DNA插入 /缺失的位置。
一个特殊实施例为， 上述多寡核苷酸引物 inter-Alu PCR产物经纯化后， 利 用"外显子捕获"技术仅分析基因区外显子中的 SNP、 点突变， 插入 /缺失与肿瘤 之间的关系。具体为，通过捕获外显子的载体 pETV— SD是一种反转录病毒穿 梭载体 (shuttle vect0r)。 因为凡是有内含子和外显子的基因在转录后都要经过 RNA剪接， 这就需要有剪接供体 (splicing donor, SDM立点和剪接受体 (splicing acceptor, SA)位点。 DNA复制子被超声打断成小片段后， 克隆在载体位于"外 显子捕获序列"下游的克隆位点上。 而后， 将这些重组载体汇总后感染反转录 病毒的专宿包装细胞系 (ecotropicretroviral packaging cell line) ψ2细胞系。
Ψ2细胞提供蛋白质产物使载体（自身不能合成病毒蛋白质)成为反转录病毒在 细胞里增殖。 当反转录病毒在细胞内转录时， 如果插入片段中包含有功能的 SA位点， 则有可能发生 RNA剪接反应而将 IVS切除。 已剪接和未剪接的病 毒 RNA都包装在病毒子 (virion)中，从细胞培养液中收集后用来感染兼宿反转 录病毒包装细胞系 (ampho tropic retroviral packagingcell line)PA-317。这使反转 录病毒再进行一轮复制， 并产生能感染猴肾细胞系 COS细胞的高效价病毒原 种。 从第二个细胞系 PA-317 细胞中分离得到的病毒， 用来感染组成型产生 SV40T(肿瘤)抗原的 COS 细胞。 病毒 RNA基因组被反转录， 并在载体上的 SV40复制起点作用下， 以环状 DNA附加体形式进行复制。从 COS细胞中回 收复制的附加体 DNA， 经限制性内切酶 Dpn l 酶切后转化细菌。 在含卡那霉 素 (Kn)和 5—氯一 4一溴一 3—吲哚一 β— D—半乳糖苷 (X-gal)的培养基上挑选 转化子。 卢一半乳糖苷酶可水解 X— gal而生成蓝色产物。 因此， 不产生 β— 半乳糖苷酶的转化子菌落则呈白色。只挑选出白色菌落作进一步研究的材料。 准确的剪接反应可切除作为标记的 IVS, 使人口一珠蛋白基因的第 1 外显子 与落入了捕获陷阱的插入片段中的外显子序列连接。 这样通过高通量测序技 术可以检测用于分析肿瘤基因组基因区外显子中的 SNP、 点突变， 插入 /缺失与 肿瘤的关系。 同样可借助小片段寡核苷酸分析软件包 （SOAPalinger) 的组装， 形成长链 DNA序列， 并通过对比人类基因组序列确定检测出的各 DNA序列的 位置。 另外， 各序列通过 BLAST及 UCSC数据库比对人类基因组序列， 确定各 序列中的 SNP在肿瘤与对照基因组 DNA中的差别， 点突变的位置， 也可以确定 肿瘤组织 DNA插入 /缺失的位置。
实施例 3
该实施例与实施例 1及实施例 2也有相似性。因为绝大多数的 5-甲基胞嘧啶 (5mC) 主要集中于富含 CpG位点的重复序列 Alu家族中， 并且据估计， 在整 个人类基因组， Alu家族可包含多达 33 %的 CpG位点。 既往研究表明， 在肿瘤 基因组中， Alu序列中的某些特殊 CpG位点甲基化水平出现显著性降低， 这一 现象在 AluY亚家族中尤为突出。此外，在精神分裂症的相关研究中，我们发现， AluY序列内及其周围区域的某些 CpG位点甲基化水平亦出现显著性改变。因此， 在本实施例中， 为研究各类疾病中基因区 CpG位点甲基化水平改变程度， 需要 对所选取的 AluY寡核苷酸引物的位置进行调整。 因为 DNA在经过亚硫酸氢盐 预处理后， 未被甲基化的 CpG位点中的 "C"会转换成 "T"， 所以核苷酸引物 不能包括 CpG位点， 并且需要将所选取位置中的非 CpG位点中的 "C"转换为 "T"。 另外， 为了包含更多的 Alu区域序列， 所选择的 AluY共同序列需更靠近 共同序列的中间区域。 如图 9中显示， 选取 AluY共同序列中靠近中间区域的两 段不含 CpG位点的序列作为引物。命名其中一条寡核苷酸引物为 CH11,其扩增 方向朝向 "5'"， 长度为 llbp。 该寡核苷酸引物可以进行 "头对头"方向扩增， 即一个 Alu "5， "端与另一个 Alu "5， "端之间的 DNA序列。 命名另一条寡 核苷酸引物为 CT11, 其扩增方向朝向 " 3'"， 长度为 llbp。 该寡核苷酸引物可以 进行 "尾对尾 "方向扩增， 即一个 Alu "3' "端与另一个 Alu "3' "端之间的 DNA序列。 CH11和 CT11两个寡核苷酸引物作用于同一个 inter-Alu PCR反应， 可以扩增一个 Alu "5' "端与另一个 Alu"3' "端之间的 DNA序列（"头对尾" 方向）， 以及一个 Alu "3' "端与另一个 Alu "5， "端之间的 DNA序列 （"尾 对头"方向）。
因为经亚硫酸氢盐预处理后的非甲基化" C"全部转换成" T"。所以，将设计的 2条寡核苷酸引物 CH11与 CT11中的" C"全部转换为" Τ"。图 10中所示的是 2个 AluY共同序列寡核苷酸引物中 "C"变 "T" 的具体位置。 2个 AluY共同序列 寡核苷酸引物经过将 "C" 转换成 "T" 后， 它们的序列分别是： CH11 "5' TTTAATAAAAA3'", CT11 " 5 ' AACATCAAAAT 3 "Ό 图 11与图 6显示的 3个 寡核苷酸引物的 inter-Alu PCR扩增方向相同， 所得 PCR产物依据 Alu寡核苷酸 引物扩增的方向性，可包括一个 Alu"3' "端与另一个 Alu"3' "端之间的 DNA 序列 （"尾对尾"方向）， 一个 Alu "5' "端与另一个 Alu "5 ' "端之间的 DNA 序列 （"头对头"方向）； 一个 Alu "5' "端与另一个 Alu"3' "端之间的 DNA 序列 （"头对尾"方向） 以及一个 Alu "V "端与另一个 Alu "5' "端之间的 DNA序列 （"尾对头"方向）。 所示实施例中， 2个不同方向的 AluY共同序列寡 核苷酸引物扩增经亚硫酸氢盐预处理后的肿瘤组织和对照组织（包括外周血）基 因组 DNA， 可产生上述 4种不同的扩增产物。 如图 11所示， 扩增产物包括 "头 对头"、 "尾对尾"、 "头对尾"及"尾对头 "4个方向的 DNA复制子。 经 DNA琼脂 糖凝胶电泳检测， 扩增产物显示为白色斑片影。 具体为， 取 900ng基因组 DNA 加入 0.3M氢氧化钠（NaOH)并放置于 42°C， 持续 20分钟。 之后在 95 °C放置 3 分钟， 0°C放置 1分钟。 此后， 上述样品加入 PH为 5.0的焦亚硫酸钠 （2.0M) 及对苯二酚 （0.5mM) ， 上层覆盖矿物油， 放置于 55 °C， 持续 16小时。 经亚硫 酸氢盐预处理后的 DNA经过纯化， 可通过 inter-Alu PCR反应进行扩增。 每个 PCR 反应体系的总体积为 20μ1， 包括 5 x Mastermix (10 x PCR 扩增缓冲液
( 500mM KC1， lOOmM Tris-Cl, 15mM MgCl2)， 50mM MgCl2，及 lOmM 4禾中 dNTP 混合液）4 l，5uM 引物 CH11 l l，5uM 引物 CT11 Ιμΐ，热稳定 DNA聚合酶 Ο.ΐμΐ, lOng/μΙ经亚硫酸氢盐预处理后的基因组 ϋΝΑ 2μ1， 及去离子水 11.9μ1。 PCR扩 增反应包括： 95 °C变性 5分钟， 而后 20个扩增循环又包括 95 °C变性 30秒， 52 °C 退火 30秒， 72°C延伸 2分钟； 最后 72°C 延伸 5分钟终止反应。 因为经过亚硫 酸氢盐预处理过的基因组 DNA为单链 DNA， 所以在 inter-Alu PCR反应中不容 易扩增。因此需要重复进行上述 inter-Alu PCR反应，以增加 DNA复制子的质量。 反应完毕后， 取 5 L与 50 %甘油混合后进行 1.5 %琼脂糖凝胶电泳， 在紫外灯下 观察扩增产物的质量。 所获得的 DNA复制子是集中于基因区 Alu及 Alu周围区 域的 DNA片段。 在图 12中， 新一代测序技术仅需要 3 gPCR产物， 就可以检 测到经亚硫酸氢盐预处理后的 DNA序列， 甲基化的 "C"不会产生变化， 而非 甲基化的 " C " 会转变成 T。 此后， 经过前述小片段寡核苷酸分析软件包
( SOAPalinger)的组装， 形成长链 DNA序列， 并通过对比人类基因组序列确定 检测出的各 DNA序列的位置。另外， 各序列通过 BLAST及 UCSC数据库比对 人类基因组序列， 确定各序列中基因区肿瘤与对照基因组 DNA CpG位点中的
"C" 甲基化程度是否存在差别。
上述实施例 3亦可用于对其他疾病的甲基化水平改变的探索， 也就是说， 虽 然在实施例 1中， 高通量测序仪器的测序结果显示， 58 %的基因区序列所属基因 为肿瘤相关基因， 但是所有的基因均存在与各类疾病相关的可能性， 因此， 该方 法可以扩展到对其他疾病基因区特征的研究中。

Claims

权 利 要 求 书
1. 以 Alu间聚合酶链式反应为基础的检测基因区特征的方法， 包括以下步骤：
(1)以 Alu家族共同序列为主要寡核苷酸引物，利用单一或多寡核苷酸引物作 为引物对样本 DNA进行 inter-Alu PCR扩增；
(2)采用循环阵列合成测序法为基础的高通量测序仪器进行测序；
(3)对测序结果进行基因组中基因区单核苷酸多态性 （SNP)、 点突变， 插入 / 缺失及双脱氧核苷酸分子 （DNA) 甲基化 CpG位点的检测。
2. 根据权利要求 1所述的方法， 其特征是： 所述被扩增的样本 DNA为多个两 Alu 序列间的 DNA片段。
3. 根据权利要求 1所述的方法， 其特征是： 所述步骤 (1)的样本 DNA是利用苯 酚 /氯仿法提取组织或外周血分离的白细胞中的 DNA， 提取后的 DNA使用 琼脂糖凝胶电泳， 通过溴乙锭染色， 在紫外灯下观察 DNA提取质量。
4. 根据权利要求 1所述的方法， 其特征是： 以 AluY共同序列中的片段设计的 寡核 苷 酸 弓 I 物 ， 其序 歹 'J 为 5 ' -GAGCGAGACTCCGTCTCA-3 ' ， 5' -TGGTCTCGATCTCCTGACCTC-3或 5 ' -TGGTCTCGATCTCCTGAC CTC-3' 。
5. 根据权利要求 1所述的方法， 其特征是： 以 AluY共同序列寡核苷酸引物及 Alu共同序列引物 R12A/267的混合物作为引物， 以扩增更多基因区序列。
6. 根据权利要求 1所述的方法，其特征是：所述 inter-Alu PCR使用热稳定 DNA 聚合酶。
7. 根据权利要求 1所述的方法， 其特征是： 在进行 DNA CpG位点甲基化水平 的检测时， DNA需要亚硫酸氢盐的处理。
8. 根据权利要求 7所述的方法， 其特征是： inter-Alu PCR以 AluY共同序列中 靠近中间区域的两段不含 CpG位点的序列作为引物， 弓 I物 CH11朝向 "5' " 方向扩增， 长度为 llbp， 序列为 "5' TTTAATAAAAA 3' "; CT11朝向 " 3' " 方向扩增， 长度为 llbp， 序列为 "5' AACATCAAAAT 3' "。
9. 根据权利要求 7所述的方法， 其特征是： 在进行 DNA CpG位点甲基化水平 的检测时， 新一代测序技术检测对比经亚硫酸氢盐处理后的肿瘤及非肿瘤基 因组 DNA甲基化水平差异， 主要检测区域为富含 CpG位点的基因组 Alu区 域及 Alu旁区域。
10. 根据权利 7所述的方法， 其特征是： 以 AluY或 Alu共同序列中任意位置的 不含 CpG位点的序列经过 "C"转换为 "T"后， 作为寡核苷酸引物。
PCT/CN2011/072204 2010-03-30 2011-03-28 以alu间聚合酶链式反应为基础的检测基因区特征的方法 WO2011120409A1 (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115410649A (zh) * 2022-04-01 2022-11-29 北京吉因加医学检验实验室有限公司 一种同时检测甲基化和突变信息的方法及装置

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101792808A (zh) * 2010-03-30 2010-08-04 广州市香港科大霍英东研究院 以Alu间聚合酶链式反应为基础的检测基因区特征的方法
CN102134610B (zh) * 2010-12-15 2013-04-10 山东大学齐鲁医院 重型肝炎相关基因启动子甲基化状态检测的方法及试剂盒
CN102912010B (zh) * 2012-06-25 2014-07-23 海南广陵高科实业有限公司 一种快速、准确的检测杂交水稻种子纯度的方法
EP3172342A4 (en) 2014-07-25 2017-12-13 GE Healthcare UK Limited Screening and monitoring the progression of type 2 diabetes by the molecular identification of gut flora using fta as a faecal collection device
CN104498591B (zh) * 2014-11-24 2016-06-29 南通大学附属医院 基于液相芯片定量检测Alu基因甲基化水平的方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5773649A (en) * 1996-06-10 1998-06-30 Centre De Recherche De L'hopital Sainte-Justine DNA markers to detect cancer cells expressing a mutator phenotype and method of diagnosis of cancer cells
US7537889B2 (en) * 2003-09-30 2009-05-26 Life Genetics Lab, Llc. Assay for quantitation of human DNA using Alu elements
CN101792808A (zh) * 2010-03-30 2010-08-04 广州市香港科大霍英东研究院 以Alu间聚合酶链式反应为基础的检测基因区特征的方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NL9100132A (nl) * 1991-01-25 1992-08-17 Ingeny Bv Werkwijze voor het detecteren van dna sequentievariatie.
US20060019270A1 (en) * 2004-04-01 2006-01-26 Board Of Regents The University Of Texas System Global DNA methylation assessment using bisulfite PCR

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5773649A (en) * 1996-06-10 1998-06-30 Centre De Recherche De L'hopital Sainte-Justine DNA markers to detect cancer cells expressing a mutator phenotype and method of diagnosis of cancer cells
US7537889B2 (en) * 2003-09-30 2009-05-26 Life Genetics Lab, Llc. Assay for quantitation of human DNA using Alu elements
CN101792808A (zh) * 2010-03-30 2010-08-04 广州市香港科大霍英东研究院 以Alu间聚合酶链式反应为基础的检测基因区特征的方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115410649A (zh) * 2022-04-01 2022-11-29 北京吉因加医学检验实验室有限公司 一种同时检测甲基化和突变信息的方法及装置

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