WO2004018691A1 - Procede pour adn chromosomique micro-excise d'amplification et son utilisation - Google Patents

Procede pour adn chromosomique micro-excise d'amplification et son utilisation Download PDF

Info

Publication number
WO2004018691A1
WO2004018691A1 PCT/CN2002/000580 CN0200580W WO2004018691A1 WO 2004018691 A1 WO2004018691 A1 WO 2004018691A1 CN 0200580 W CN0200580 W CN 0200580W WO 2004018691 A1 WO2004018691 A1 WO 2004018691A1
Authority
WO
WIPO (PCT)
Prior art keywords
alu
dna
sequence
primers
specific primers
Prior art date
Application number
PCT/CN2002/000580
Other languages
English (en)
French (fr)
Inventor
Xinyuan Guan
Janothan S. T. Sham
Liang Hu
Original Assignee
The University Of Hong Kong
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The University Of Hong Kong filed Critical The University Of Hong Kong
Priority to PCT/CN2002/000580 priority Critical patent/WO2004018691A1/zh
Priority to CN02827308.7A priority patent/CN1288252C/zh
Publication of WO2004018691A1 publication Critical patent/WO2004018691A1/zh

Links

Classifications

    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a method for amplifying micro-cut chromosomal DNA.
  • the amplified product obtained by this new method contains substantially no genomic repeats.
  • the FISH probe prepared using the amplified product can effectively eliminate non-specific hybridization signals caused by repeated sequences, thereby improving the accuracy of the FISH probe. Background technique
  • Chromosome microdissection technology is a cell and molecular biology technology established in the 1980s. In the early 1990s, Meltzer et al. Combined it with chromosomal fluorescence in situ hybridization to create micro-fluorescence in situ hybridization (Micro- FISH) (Meltzer PS, Guan X-Y, Burgess A, Trent JM: Nature Genetics, 1:24, 1992) 0 Since then, after nearly ten years of efforts, Micro-FISH technology has been widely used in cell biology and In molecular biology research, it has become one of the key technologies in chromosome diagnosis.
  • Micro-FISH micro-fluorescence in situ hybridization
  • This library can be used for molecular targeting cloning (GuanX-Y, Meltzer PS, Cao J, and Trent JM: Genomics, 14: 680, 1992).
  • chromosome-related staining probes such as whole chromosome staining probes (Guan XY, Meltzer PS, and Trent JM: Genomics, 22: 101, 1994), chromosome arm staining probes (Guan XY, Zhang HE, Bitter M, Jiang Y, Meltzer PS, and Trent JM: Nature Genetics, 12:10, 1996), chromosome band-specific probes (Guan XY, Meltzer PS, Burgess A, and Trent JM: Human Genetics, 95: 637, 1995), etc., such probes have been widely used in the study of chromosomal mutations and congenital aberrations in solid tumors, and have been used for prenatal diagnosis.
  • the traditional microscopy fluorescence in situ hybridization process is complicated and very easy to be contaminated with exogenous DNA, so the probability of success is not high. It mainly includes two parts, (a) chromosome microdissection and (b) PCR amplification of the cut DNA fragment.
  • the specific steps are as follows: 1) Use a thin glass needle to cut the chromosome fragment to be cut seen by the microscope, and then transfer the cut DNA fragment to a 0.5 ml small test tube, in which 5 Microliters of PCR amplification reagents. Generally, 5-20 copies of DNA fragments need to be cut. 2) The DNA is treated with Topo I enzyme, and the DNA is double-stranded uncoiled (U.S.
  • Patent 5,545,524) or the DNA is pretreated with pepsin (U.S. Patent 6,228,587), and then amplified with a PCR primer called UN1 Increased DNA (CCGACTCGAGNNNNNNATGTGG, SEQ ID NO: 3), UNI contains 22 bases, of which the first 6 bases are specific sequences, and the six from 7 to 12 are random combinations, so this primer only needs the first 6 bases Corresponding to the amplified DNA, it can be paired with the 12 bases in the DAN sequence to amplify the DNA.
  • the main advantage of this primer is its low specificity, which can amplify various DNAs.
  • UN1 also has many shortcomings: 1) Due to the lack of specificity, the primer can not only amplify human DNA that is cut, but also other DNA including bacteria, because the amount of DNA that is cut is small, so any Exogenous DNA contamination can cause experiments to fail. 2) Since the substance has only 12 specifically paired bases, the temperature required for pairing with denatured cleaved DNA is very low, generally around 30 ° C, and 56 D C required for general PCR. The difference is very large, so one step of pre-PCR must be added. This step is very tedious, and it is easy to cause exogenous DNA contamination when it is performed, making the entire experiment fail.
  • the primer Since 6 of the 22 bases of the primer are random, the primer actually contains 4096 (4 6 ) different combinations of primers, and the concentration of each of them is not high . Due to the above-mentioned major disadvantages, the use of this technology is greatly restricted.
  • the DNA amplified by the UN1 primer contains a large number of repeats. These repeats will non-specifically hybridize with a large number of corresponding repeats scattered in the human genome when in situ hybridization, thereby increasing the background signal of hybridization. .
  • pre-hybridization with unlabeled human repeats (such as Alu sequences, satellite sequences, etc.) must be performed to prevent these labeled repeats from hybridizing with repeats in the genome.
  • Alu sequence appears most frequently, and appears approximately every 4 kilobase pairs.
  • the Alu sequence accounts for about 5% of the total DNA of the human genome, up to 900,000 copies, of which 60% of the sequence members contain the cut of the restriction enzyme Alu, so it is called the Alu family, which is mainly concentrated in the R band in the late stage of cell division. It belongs to non-coding DNA, but some of it is located in the untranslated region of raRNA, even in the coding region.
  • the longest Alu sequence is about 150 base pairs.
  • the human Alu sequence is about 300bp in length, and the body is composed of repeating sequences of 120bp and 150bp. The two are separated by A-rich regions, and there is a 7-10bp forward repeat at both ends. Alu sequences are not exactly the same, there are some differences. These repeats can interfere with specific hybridization signals because they can hybridize to each other to generate non-specific background signals. This is currently the biggest challenge in making FISH probes from microdissected chromosomal DNA.
  • FISH probes can be used for a variety of purposes, including genetic disease diagnosis (mainly various chromosomal abnormalities), prenatal diagnosis (pregnancy genetic disease screening), tumor typing, Diagnostic and prognostic tests, observation of radiation and other environmental factors to human damage, therefore, there is an urgent need in the art to improve the micro-fluorescence in situ hybridization technology in order to obtain FISH probes with reduced non-specific background signals.
  • the purpose of the present invention is to provide a new method for amplifying microscopically cut chromosomal DNA and its application in preparing FISH.
  • the method can effectively remove repeated sequences, thereby eliminating non-specific hybridization signals caused by repeated sequences. Interference, improving the accuracy and reliability of hybridization results.
  • a method for amplifying micro-cut chromosomal DNA is provided, which comprises the steps of: performing an amplification reaction by using the micro-cut chromosomal DNA as a template and Alu-specific primers as primers. ,
  • the Ahi-specific primers specifically bind to the 5 'end of the Alu sequence and extend in the 3'-5' direction of the Alu sequence, or all bind to the 3 'end of the Alu sequence and extend in the Alu sequence. 5 ' ⁇ 3' direction.
  • the amplification reaction is a polymerase chain reaction.
  • the length of the Alu-specific primer is 15-25 bp.
  • the Alu-specific primer is selected from the following group ⁇
  • AD-1 5'- ACA GAG YRA GAC TCY RTC TCA AC -3 '(SEQ ID N0: 1)
  • a method for generating a fluorescently labeled probe which includes steps:
  • a polynucleotide product is amplified by an amplification reaction in which microscopically cut chromosomal DNA is used as a template and Alu-specific primers are used as primers, wherein the Alu-specific primers are all Specifically bind to the 5 'end of the Alu sequence and extend in the 3' ⁇ 5 'direction of the Alu sequence, or both bind to the 3' end of the Alu sequence and extend in the 5'-3 'direction of the Ahi sequence;
  • step (b) Fluorescently labeling the polynucleotide product in step (a) to generate a fluorescently labeled probe.
  • the fluorescently labeled probe is a fluorescent in situ hybridization probe.
  • a fluorescently labeled probe prepared by the above method is also provided.
  • Figure 1 is an electrophoresis image of a PCR product amplified with Alu primers, where the molecular weight marker (SM) is a 100 bp DNA ladder. Lane 1 indicates that no DNA template is added; Lane 2 indicates that AD-2 is used as a primer, and micro-cut DNA is used as a template; Lane 3 is used that uses AD-2 as a primer, and the whole genome DNA is used as a template.
  • SM molecular weight marker
  • Fig. 2 is a fluorescence in situ hybridization (FISH) image of a fluorescently labeled probe prepared by the above method.
  • FISH fluorescence in situ hybridization
  • the term "microscopically cut chromosomal DNA fragment” refers to a chromosome fragment obtained by microdissection, which is not particularly limited in length, and is usually 5,000 to 20,000 kb.
  • the term "Alu-specific primer” refers to a primer that specifically binds to the 3 'terminus of the Alu sequence such that the polymerase chain reaction amplification product does not contain or does not substantially contain the Alu repeat sequence.
  • Alu-specific primers are divided into two types: The first type of Alu-specific primers specifically bind to the 5 'end of the Alu sequence and extend in the 3'-5' direction of the Alu sequence; the second type of Alu-specific primers are Binding to the 3 'end of the Alu sequence and extending in the 5'-3' direction of the Alu sequence.
  • the inventors After extensive and in-depth research, the inventors have established a new method for efficiently amplifying DNA sequences using micro-cut chromosomal DNA as a template.
  • This method uses a uniquely designed amplification primer (ie, Alu-specific primer) to replace the UN1 primer, where the amplification primer is located at the 3 'end or 5' end of the Alu repeat that occurs at high frequency in human genomic DNA.
  • Alu-specific primer a uniquely designed amplification primer
  • the characteristic of this method is that using this primer to amplify the human genomic DNA sequence in the micro-cut chromosomal DNA by PCR method can effectively remove the high-frequency Alu repeated sequences in human genomic DNA.
  • the principle of this technical method is to use the characteristics of the frequency of Alu sequences, and use the Alu-end sequence as a template to design corresponding primers.
  • two Alu sequences are not far apart, that is, within a normal PCR reaction amplification range (generally within 2 kilobase pairs) and the directions of the two Alu sequences are opposite (such as 3 'end to 3' end)
  • the genomic DNA sequence between these two Alu sequences can be amplified. Because the DNA sequence amplified by this method is located between two Alu repeats, it no longer contains duplicates.
  • the PCR reaction conditions are not particularly limited, and conventional specific amplification PCR conditions can be used in the present invention.
  • a common condition is denaturation at 90-95 ° C for 45-75 seconds, annealing at 50-65 ° C for 30-90 seconds, and 70-74 ° extension for 30-90 seconds, a total of 25-45 cycles (preferably 30-40) Cycles).
  • AD-1 or AD-2 can only specifically amplify the DNA sequence derived from the human genome, thereby greatly reducing the amount of foreign DNA during amplification '( (Such as bacterial DNA) contamination, improving the success rate of chromosomal microdissection.
  • This method can simplify the steps of DNA amplification. It is no longer necessary to treat microscopically cut chromosomal DNA with Topo I enzyme or pepsin, and there is no need to add another step of pre-PCR, thereby reducing the need for chromosomal microdissection. Time and reduced costs. (Of course, if needed, you can still pre-treat with Topo I or pepsin before PCR amplification).
  • the method When the method is used to amplify the microscopically cut chromosomal DNA, it can effectively remove the repeated sequences in the DNA, thereby eliminating the interference of non-specific hybridization signals caused by the repeated sequences.
  • the technology of the present invention has multiple uses, including but not limited to:
  • the amplified single sequences can be used as gene probes for Southern blot molecular hybridization
  • amplified single sequences can be fluorescently labeled for chromosomal fluorescence in situ hybridization (FISH);
  • the present invention also provides a method for generating a fluorescently labeled probe, especially a FISH probe.
  • the method of the present invention for preparing a FISH probe differs from the prior art only in the DNA used as a probe. Therefore, various conventional methods for preparing FISH probes and fluorescent dyes (such as fluorescein, rhodamine, etc.) can be used in the present invention.
  • the amplification product obtained by the method of the present invention eliminates the Alu repeat sequence, it is prepared by using it
  • the non-specific background signal of the FISH probe can be significantly reduced.
  • the present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention.
  • the experimental methods without specific conditions in the following examples are generally based on conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer Suggested conditions.
  • Example 1 Example 1
  • AD-2 5'- ACC AAC GAA TTC AGA CTC YRT CTC AAC —3 '(SEQ ID NO: 2) where: Y or T; 1? Or 6. Both primers specifically bind to the 3 'end of the Alu sequence and extend in the 5' ⁇ 3 'direction of the Alu sequence.
  • SEQ ID NO: 2 5'- ACC AAC GAA TTC AGA CTC YRT CTC AAC —3 '(SEQ ID NO: 2) where: Y or T; 1? Or 6. Both primers specifically bind to the 3 'end of the Alu sequence and extend in the 5' ⁇ 3 'direction of the Alu sequence.
  • Example 1 5 to 10 copies of the microdissected chromosomal DNA in Example 1 (as a template for a PCR reaction) are taken, and the reaction is performed in a total reaction volume of 50 ⁇ 1 (microliter).
  • the components in this reaction system are: 10 mM Tris-HCl, pH 8. 4, 2 mM MgCl, 50 mM KC1, 0.1 raM gelatin, 200 mM DNTP, 0.5 mM primers AD-1 or AD-2, 2 units Taq polymerase).
  • the PCR reaction conditions are: denaturation at 92-95 ° C for 45-75 seconds, annealing at 50-65 ° C for 30-90 seconds, and extension at 70-74 ° C for 60-120 seconds, for a total of 30-40 cycles.
  • PCR products were detected by gel electrophoresis. The results are shown in Fig. 1. The size of the PCR products is between 300-800 base pairs.
  • Alu-specific primers such as AD-2 primers can specifically amplify DNA sequences derived from the human genome (lane 3) without non-specifically amplifying DNA from other sources. It has also been shown that AD-2 primers can effectively amplify microdissected DNA (lane 2). Like the AD-2 primer, the AD-1 primer is also effective in amplifying microdissected chromosome legs.
  • FISH Chromosome Fluorescence In situ Hybridization
  • Chromosomal fluorescence in situ hybridization was used to detect the results of chromosomal microdissection.
  • the specific process is as follows:
  • the first PCR product was fluorescently labeled with the PCR method to generate fluorescently labeled probes.
  • the PCR reaction conditions are: denaturation at 92-95 ° C for 45-75 seconds, annealing at 50-65 ° C for 30-90 seconds, and 70-74 ° extension for 60-120 seconds, for a total of 20-30 cycles.
  • the template is the amplification product in Example 3, and the primers are AD-1 or AD-2 primers.
  • the fluorescently labeled probe was centrifuged by precipitation and dissolved in TE buffer. Each FISH reaction requires 100-200 nanograms of fluorescent probes. The probes are dissolved in 10 microliters of FISH reaction buffer. After denaturation at 75 ⁇ 10 minutes, overnight hybridization with metaphase chromosomes that also undergo denaturation ( See Guan X-Y, Trent JM, and Meltzer PS: Human Molecular Genetics, 2: 1117, 1993). After hybridization, after washing and fluorescence measurement, the hybridization results can be observed under a fluorescence microscope (see Figure 2).
  • the PCR amplification product obtained above was cloned into a plasmid vector (pBS) for storage and future replication in large quantities.
  • pBS plasmid vector
  • a restriction endonuclease EcoRI cut point (GAATTC) was designed in the AD-2 primers. After EcoRI digestion, the PCR amplification product can be cloned into EcoRI digested plasmid vector.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Description

扩增显微切割的染色体 DNA的方法及其应用 技术领域
本发明涉及一种扩增显微切割的染色体 DNA的方法。 利用该新方法得到 的扩增产物中基本上不含有基因组重复序列。利用该扩增产物制备的 FISH探 针可有效地消除了因重复序列引起的非特异性杂交信号，从而提高了 FISH探 针的准确性。 背景技术
染色体显微切割技术是 1980 年代建立起的一项细胞分子生物学技术， 1990年初， Meltzer等人将其与染色体荧光原位杂交技术相结合， 创立了显 微 -荧光原位杂交技术 （Micro- FISH) (Meltzer PS, Guan X- Y， Burgess A, Trent JM: Nature Genetics, 1:24, 1992 ) 0 此后， 经过近十年的努力， Micro-FISH技术已广泛地被用于细胞生物学及分子生物学研究中， 成为染色 体诊断中的关键技术之一。
以下是染色体显微切割技术应用较广泛的几个方面：
1. 被用来制备染色体特异区带 DNA文库。 此文库可以用来做分子定位克 隆 （GuanX—Y, Meltzer PS, Cao J, and Trent JM: Genomics, 14:680, 1992) 。
2. 被用来制备各类染色体相关涂染探针， 比如全染色体涂染探针 （Guan X-Y, Meltzer PS, and Trent JM: Genomics, 22:101, 1994) , 染色体臂涂 染探针 (Guan X-Y, Zhang HE, Bitter M， Jiang Y， Meltzer PS, and Trent JM: Nature Genetics, 12:10， 1996) ， 染色体区带特异性探针 (Guan X-Y, Meltzer PS, Burgess A, and Trent JM: Human Genetics, 95:637， 1995) 等， 此类探针已被广泛地应用于实体肿瘤染色体变异及先天性染色体畸变的 研究中， 并已被用来进行产前诊断。
3. 被用来直接检测未知来源的标记染色体 （Unknown Marker) , 以确定 该染色体的组 成及来源 （Guan X-Y, Meltzer PS, Dalton WS， Trent JM: Nature Genetics, 8:155， 1994; Guan X~Y, Horsman D， Zhang H, Meltzer PS, and Trent JM: Blood; 88:1418, 1996) 。 4. 被用来筛选染色体特异性区带扩增的癌基因相关基因 （Guan X- Y， Xu J, Anzick SL, Zhang HE, Trent JM, and Meltzer PS : Cancer Research, 56 : 3446, 1996 ; ) 。
传统的显微荧光原位杂交技术过程复杂，且非常容易被外源性 DNA污染， 所以成功的机率不高。 它主要包括二个部分， （a)染色体显微切割及（b)将切 割的 DNA片段进行 PCR扩增。 具体步骤如下： 1)用细玻璃针将显微镜所看到 的想要切割的染色体片段切割下来， 然后将被切割下的 DNA片段转移到 0. 5 毫升的小试管中， 在试管中装有 5微升的 PCR扩增试剂。 一般需要切割 5-20 拷贝的 DNA片段。 2) 用 Topo I 酶处理 DNA， 其 DNA双链 解螺旋（美国专利 5,545,524 ) , 或者用胃蛋白酶预处理 DNA (美国专利 6， 228， 587 )， 然后用一 种被称为 UN1的 PCR引物扩增 DNA (CCGACTCGAGNNNNNNATGTGG, SEQ ID NO: 3) , UNI中含有 22个碱基， 其中前 6个碱基为特异序列， 从第 7到 12 这六个为随机组合， 所以该引物只要前 6个碱基与被扩增的 DNA相对应， 就 可以与该 DAN序列中的 12个碱基配对， 从而扩增 DNA。 该引物的主要优点是 特异性低， 可以将各种 DNA扩增。 但是 UN1也存在着许多缺点： 1) 由于缺乏 特异性， 该引物不仅可以扩增被切割的人 DNA， 也可同时扩增包括细菌在内 其它 DNA, 由于被切割的 DNA量很小， 所以任何外源性 DNA污染都会导致实 验失败。 2) 由于该物仅有 12个特异相配对的碱基， 所以与变性的被切割的 DNA相配对时所要求的温度很低， 一般 30 °C左右， 与一般的 PCR所要求的 56 DC相差甚远， 所以必须加一步预 PCR, 此步骤非常繁琐， 而且在进行时容易 导致外源性 DNA污染， 使整个实验失败。 3) 由于该引物的 22个碱基中有 6 个是随机的， 所以该引物实际上包含有 4096 ( 46 ) 个 不同组合的引物， 对 其中每一种引物来说， 其浓度都不高。 由于上述几种主要缺点， 该技术的使 用受到了很大的限制。 另外， 由 UN1引物所扩增的 DNA中含有大量的重复序 列， 这些重复序列在进行原位杂交时会与散布在人基因组中的大量相应的重 复序列进行非特异性杂交， 而增加杂交的背景信号。 所以在进行染色体原位 杂交前， 必须先与未标记的人重复序列 （如 Alu序列， 卫星体序列等） 进行 预杂交， 从而阻断这些被标记的重复序列与基因组中的重复序列杂交。
在人类基因组中存在许多重复的 DNA序列。 这些序列非常相似， 在整个 基因组中不断出现。 其中， 以 Alu序列出现频率最高， 大约每隔 4千碱基对 就会出现一次。 Alu序列占人类基因组总 DNA量 5%左右， 高达 900, 000拷贝， 其中 60%序列成员含有限制性内切酶 Alu的切口， 故叫做 Alu家族， 主要集 中在细胞分裂晚期的 R带， 大部分属于非编码 DNA, 但也有一部分位于 raRNA 的非翻译区， 甚至位于编码区内。 Alu序列最长的约 150碱基对。 人类 Alu 序列长度约 300bp,本.身又由 120bp和 150bp的重复序列组成， 两者之间由富 含 A的区域分开， 两端又有一段 7- 10bp的正向重复序列。 Alu序列之间并不 完全一样， 存在一些差异。 由于这些重复序列可以相互杂交而产生非特异背 景信号， 因而会干扰特异性杂交信号。 这是目前用显微切割的染色体 DNA制 作 FISH探针所面临的最大难题。
由于荧光原位杂交（Fluorescence In Site Hybridization, FISH)探针 可用于多种用途， 其中包括遗传病诊断（主要是各种染色体异常）， 产前诊断 (孕妇遗传病筛查）， 肿瘤分型、 诊断和预后检验， 观测放射线和其他环境因 素对人体的损害， 因此， 本领域迫切需要改进显微-荧光原位杂交技术， 以便 获得非特异性背景信号降低的 FISH探针。 发明内容
本发明的目的在于提供一种新的扩增显微切割的染色体 DNA的方法及其 在制备 FISH中的应用， 该方法可以有效地去除重复序列， 从而消除因重复序 列导致的非特异性杂交信号的干扰， 提高杂交结果的准确性和可靠性。 在本发明的第一方面， 提供了一种扩增显微切割的染色体 DNA的方法， 它包括步骤： 以显微切割的染色体 DNA为模板， 并且以 Alu特异性引物为引 物， 进行扩增反应，
其中所述的 Ahi特异性引物都特异性地结合于 Alu序列的 5'端且延伸方 向为 Alu序列的 3'— 5'方向， 或者都结合于 Alu序列的 3'端且延伸方向为 Alu 序列的 5'→3'方向。
在另一优选例中， 所述扩增反应是聚合酶链反应。
在一优选例中， 所述的 Alu特异性引物长度为 15- 25bp。 在另一优选中， 所述的 Alu特异性引物选自下组-
AD-1 : 5'- ACA GAG YRA GAC TCY RTC TCA AC -3' (SEQ ID N0 : 1)
AD- 2 : 5'- ACC AAC GAA TTC AGA CTC YRT CTC AAC-3* (SEQ ID NO : 2) 其中： Y=C或 T; ！？^或 。
在本发明的第二方面， 提供了一种产生荧光标记探针的方法， 它包括步 骤：
(a) 通过扩增反应扩增出多核苷酸产物， 在所述扩增反应中以显微切割 的染色体 DNA为模板， 并且以 Alu特异性引物为引物， 其中所述的 Alu特异 性引物都特异性地结合于 Alu序列的 5'端且延伸方向为 Alu序列的 3'→5'方 向， 或者都结合于 Alu序列的 3'端且延伸方向为 Ahi序列的 5'— 3'方向；
(b)对步骤 (a)中的多核苷酸产物进行荧光标记， 产生荧光标记探针。
在本发明的一优选例中， 所述的荧光标记探针是荧光原位杂交探针。 在本发明的第三方面， 还提供了用上述方法制得的荧光标记探针。 附图说明
图 1是用 Alu引物扩增的 PCR产物的电泳图， 其中分子量标记物（SM)是 100 bp的 DNA梯（DNA ladder)。 泳道 1表示不加任何 DNA模板； 泳道 2表 示以 AD-2为引物， 以显微切割的 DNA为模板； 泳道 3表示以 AD- 2为引物， 以全基因组 DNA为模板。
图 2是用上述方法制得的荧光标记探针所作的荧光原位杂交（FISH)图。 以 AD-2为引物， 用 PCR方法荧光标记第一轮 PCR产物，产生荧光标记探针。 图 2A表示以 AD- 2为引物， 以显微切割的 DNA为模板制得的 17号全染色体荧 光标记探针 （红色信号） ，其中 chromosome 17表示 17号染色体。 图 2B表示 以 AD-2为引物，以显微切割的 DNA为模板制得的 l lql3 HSR荧光标记探针（红 色信号） 。 具体实施方式
如本文所用， 术语 "显微切割的染色体 DNA片段" 指通过显微切割术而 获得的染色体片段， 其长度没有特别限制， 通常为 5， 000— 20， 000 kb。 如本文所用， 术语 "Alu特异性引物" 指特异性结合于 Alu序列的 3'端 末端， 从而使聚合酶链反应扩增产物不含有或基本上不含有 Alu重复序列的 引物。 Alu特异性引物分为二类： 第一类 Alu特异性引物是特异性地结合于 Alu序列的 5'端且延伸方向为 Alu序列的 3'— 5'方向； 第二类 Alu特异性引物 是结合于 Alu序列的 3'端且延伸方向为 Alu序列的 5'— 3'方向。 本发明人经过广泛而深入的研究， 建立了一种以显微切割的染色体 DNA 为模板， 高效扩增 DNA序列的新方法。 该方法采用了设计独特的扩增引物（即 Alu特异性引物）来替代 UN1引物， 其中扩增引物在位于人基因组 DNA中高频 率出现的 Alu重复序列的 3'端或 5'端。 该方法的特点是， 利用这种引物通过 PCR方法扩增显微切割的染色体 DNA中的人基因组 DNA序列， 可以有效地去 除人基因组 DNA中高频率出现的 Alu重复序列。
本技术方法的原理是利用 Alu序列出现频率的特点， 以 Alu—端的序列 为模板， 设计相应的引物。 当两个 Alu序列相隔不远， 即在一个正常 PCR反 应扩增范围内（一般在 2千碱基对内）并且这两个 Alu序列的方向相对（如 3' 端对 3'端）时， 就可以扩增出在此两个 Alu序列之间的基因组 DNA序列。 由于 用本方法扩增出的 DNA序列位于两个 Alu重复序列之间， 所以它不再含有重 复序列。
以大小为约 5000千碱基对的、显微切割的染色体 DNA片段为例， 以平均 每 4千碱基对出现一次 Alu序列计算，通常在一个片段中可以有 1250个左右 的 Alu序列， 其中相当一部分相隔距离在 PCR反应扩增范围内。 因此， 当使 用本发明的 Alu特异引物扩增该显微切割的 DNA片段中的人基因组 DNA时， 就可以大大增加得到单一序列的机会。
在本发明中， 对 PCR反应条件没有特别限制， 常规的特异性扩增的 PCR 条件都可用于本发明。 一种常用的条件是 90- 95°C变性 45-75秒， 50-65°C退 火 30-90秒， 70- 74Ό延伸 30-90秒，共 25-45个循环（较佳地 30-40个循环）。
实验结果证明了此新技术方法的可行性和高效率。以例如 5- 10个显微切 割的染色体 DNA片段为模板， 分别用本发明设计的 Alu序列 3'端特异引物扩 增时， 所有的 PCR都得到多种 PCR产物。 因此， 利用本发明的新技术方法可以快速从显微切割的染色体 DNA中扩 增出基本上不含重复序列的单一人基因组 DNA序列。 与传统的用 腿 引物引物扩增被显微切割的人染色体 DNA 相比较， 用 AD - 1或 AD- 2引物扩增被显微切割的人染色体 DNA具有以下三个主要优点：
1) 由于 Alu序列是人基因组中特异性重复序列， 因此， AD- 1或 AD- 2只 能特异性地扩增来源于人基因组的 DNA 序列， 从而大大减少扩增时被外源 DNA' (如细菌 DNA)污染的可能性， 提高染色体显微切割的成功率。
2) 该方法可以简化扩增 DNA的步骤， 不再需要用 Topo I酶或胃蛋白酶 处理被显微切割的染色体 DNA, 也不再需要另加一步预 PCR， 从而减少了染 色体显微切割所需的时间并减少了成本。 （当然， 如果需要， 仍然可以在 PCR 扩增之前用 Topo I酶或胃蛋白酶进行预处理）。
3)该方法在扩增被显微切割的染色体 DNA时， 可以有效地去除 DNA中的 重复序列， 从而消除因重复序列导致的非特异性杂交信号干扰。 本发明的技术有多种用途 ， 其中包括但并不限于：
(1) .这些被扩增得到的单一序列可以作为基因探针， 进行 Southern 印 迹分子杂交；
(2) .这些被扩增得到的单一序列可以经过荧光标记， 进行染色体荧光原 位杂交（FISH) ;
(3) .这些被扩增得到的单一序列可以结合在固相载体表面制成基因组 DNA芯片， 进行基因检测和诊断。 用本发明方法获得的多核苷酸产物或其片段可以用于制备探针， 例如染 色体荧光原位杂交 (FISH)探针。 因此， 本发明还提供了一种产生荧光标记探 针， 尤其是 FISH探针的方法。 本发明制备 FISH探针的方法与现有技术的区 别仅在于用作探针的 DNA不同。 因此， 常规的各种制备 FISH探针的方法和荧 光染料（如荧光素、 罗丹明等）都可用于本发明。
由于用本发明方法获得扩增产物消除了 Alu重复序列， 因此， 用其制备 的 FISH探针的非特异性背景信号可显著降低。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说 明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方 法， 通常按照常规条件如 Sambrook等人， 分子克隆： 实验室手册（New York : Cold Spring Harbor Laboratory Press, 1989)中所述的条件， 或按照制造厂 商所建议的条件。 实施例 1
引物合成
用人工合成的方法分别合成如下引物-
AD-1 : 5,-ACA GAG YRA GAC TCY RTC TCA AC -3* (SEQ ID N0 : 1)
AD-2 : 5'- ACC AAC GAA TTC AGA CTC YRT CTC AAC —3' (SEQ ID NO : 2) 其中： Y 或 T; 1? 或6。 . 这两个引物都特异性地结合于 Alu序列的 3'端且延伸方向为 Alu序列的 5'→3'方向。 实施例 2
染色体显微切割
在显微镜下找到要切割的染色体， 用显微操作器控制的细玻璃针切割相 应的染色体片段， 将被切割的染色体 DNA转入含有 AD- 1或 AD-2引物的 PCR 反应液中， 一般一次显微切割需要切割 5- 10个拷贝的染色体片段。 当显微切 割完成后， 被切割的染色体 DNA用于 PCR扩增反应。 实施例 3
PCR扩增
本实施例中， 取实施例 1中取 5- 10个拷贝显微切割的染色体 DNA (作为 PCR反应的模板）， 在 50 μ 1 (微升）反应总体积中进行反应。 该反应体系中的 成分为: 10 mM Tris-HCl, pH 8. 4, 2 mM MgCl, 50 mM KC1, 0. 1 raM 明胶， 200 mM DNTP, 0. 5 mM 引物 AD- 1或 AD- 2, 2 单位 Taq polymerase)。 所述的 PCR反应条件是： 92— 95°C变性 45— 75秒， 50-65°C退火 30— 90 秒， 70-74°C延 伸 60— 120秒， 共 30— 40个循环。
通过凝胶电泳来检测 PCR产物。 结果如图 1所示， PCR产物的大小分布 在 300-800个碱基对之间。 此外， PCR结果还显示 Alu特异性引物如 AD- 2引 物可以特异性地扩增来源于人基因组的 DNA序列 （泳道 3 ) ， 而不会非特异 性地扩增其他来源的 DNA。 同时也显示 AD- 2引物可以有效地扩增显微切割的 DNA (泳道 2)。 与 AD- 2引物一样， AD-1引物也能有效扩增显微切割的染色体 腿。 实施例 4
染色体荧光原位杂交（FISH)
通过染色体荧光原位杂交来检测染色体显微切割的结果。 其具体过程如 下：
用 PCR方法荧光标记第一轮 PCR产物，产生荧光标记探针。所述的 PCR 反应条件是： 92— 95°C变性 45— 75秒， 50-65°C退火 30— 90秒， 70-74Ό延伸 60— 120秒， 共 20— 30个循环。 其中的模板是实施例 3中的扩增产物， 引物 是 AD- 1或 AD - 2引物。
荧光标记探针经沉淀离心后， 溶于 TE缓冲液中。 每一次 FISH反应需要 100- 200纳克的荧光探针， 探针被溶于 10微升的 FISH反应缓冲液中， 经过 在 75Ό十分钟变性后， 与也经过变性的分裂中期染色体进行过夜杂交 （参看 Guan Χ-Υ, Trent JM, and Meltzer PS : Human Molecular Genetics, 2 : 1117, 1993 ) 。 杂交后经冲洗及荧光测定处理， 即可在荧光显微镜显微下观察杂交 结果（见图 2)。 实施例 5
对 PCR产物的保存、 筛选和鉴定
， 将上述获得的 PCR扩增产物克隆到质粒载体 (pBS)中，以便于保存及今后 大量复制。 在 AD-2引物中设计了一个限制性内切酶 EcoRI切点（GAATTC)，经 EcoRI消化后， 可以将 PCR扩增产物克隆到 EcoRI消化的质粒载体中.
然后， 用 Alu序列筛选阴性克隆。 即用含多种人基因组重复序列的细菌 00580 人工染色体（BAC)克隆 RPII-110- 0-7 (大小为 120kb)筛选出不含 Alu重复序列 的阴性克隆。 接着， 用 PCR反应扩增出插入片段， 并通过凝胶电泳法测定插 入片段的大小。 此外， 还可以进一步对一些 PCR产物做了 DNA序列测定分析。

Claims

权 利 要 求
1.一种扩增显微切割的染色体 DNA的方法， 其特征在于， 包括步骤： 以 显微切割的染色体 DNA为模板， 并且以 Alu特异性引物为引物， 进行扩增反 应，
其中， 所述的 Alu特异性引物都特异性地结合于 Alu序列的 5'端且延伸 方向为 Alu序列的 3'— 5'方向， 或者都结合于 Alu序列的 3'端且延伸方向为 Alu序列的 5'→3'方向。
2.如权利要求 1 所述的方法， 其特征在于， 所述扩增反应是聚合酶链反 应。
3.如权利要求 2所述的方法， 其特征在于， 所述的 PCR反应条件是： 90 一 95Ό变性 45— 75秒， 50-65°C退火 30— 90秒， 70-74 °C延伸 30— 90秒， 共 25— 45个循环。
4.如权利要求 1所述的方法， 其特征在于， 所述的 Alu特异性引物长度 为 15-25bp。
5.如权利要求 1所述的方法， 其特征在于， 所述的 Alu特异性引物选自 下组：
AD-1： 5'-ACA GAG YRA GAC TCY RTC TCA AC - 3' (SEQ ID N0 : 1)
AD- 2 : 5'-ACC AAC GAA TTC AGA CTC YRT CTC AAC - 3' (SEQ ID NO : 2) 其中： Y=C或 T; 1 =八或0。
6.—种产生荧光标记探针的方法， 其特征在于， 它包括步骤-
(a) 通过扩增反应扩增出多核苷酸产物， 在所述扩增反应中以显微切割 的染色体 DNA为模板， 并且以 Alu特异性引物为引物， 其中所述的 Alu特异 性引物都特异性地结合于 Alu序列的 3'端且延伸方向为 Alu序列的 5'— 3'方 向。
(b)对步骤 (a)中的多核苷酸产物进行荧光标记， 产生荧光标记探针。
7.如权利要求 6所述的方法， 其特征在于， 所述的荧光标记探针是荧光 原位杂交探针。
8.如权利要求 6所述的方法， 其特征在于， 所述扩增反应是聚合酶链反 应。
9.如权利要求 6所述的方法， 其特征在于， 所述的 Alu特异性引物长度 为 15-25bp。
10.如权利要求 6所述的方法， 其特征在于， 所述的 Ahi特异性引物选自 下组：
AD-1: 5'- ACA GAG YRA GAC TCY RTC TCA AC - 3'(SEQ ID N0:1)
AD-2: 5'-ACC AAC GAA TTC AGA CTC YRT CTC AAC-3' (SEQ ID NO :2) 其中： Y-C或 T; 1-入或0。
PCT/CN2002/000580 2002-08-22 2002-08-22 Procede pour adn chromosomique micro-excise d'amplification et son utilisation WO2004018691A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN2002/000580 WO2004018691A1 (fr) 2002-08-22 2002-08-22 Procede pour adn chromosomique micro-excise d'amplification et son utilisation
CN02827308.7A CN1288252C (zh) 2002-08-22 2002-08-22 扩增显微切割的染色体dna的方法及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2002/000580 WO2004018691A1 (fr) 2002-08-22 2002-08-22 Procede pour adn chromosomique micro-excise d'amplification et son utilisation

Publications (1)

Publication Number Publication Date
WO2004018691A1 true WO2004018691A1 (fr) 2004-03-04

Family

ID=31892709

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2002/000580 WO2004018691A1 (fr) 2002-08-22 2002-08-22 Procede pour adn chromosomique micro-excise d'amplification et son utilisation

Country Status (2)

Country Link
CN (1) CN1288252C (zh)
WO (1) WO2004018691A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992013101A1 (en) * 1991-01-25 1992-08-06 Ingeny B.V. Method of detecting dna sequence variation
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
WO2000022164A1 (fr) * 1998-10-15 2000-04-20 Genset Sondes fluorescentes de peinture chromosomique
WO2000024935A2 (en) * 1998-10-26 2000-05-04 Yale University Allele frequency differences method for phenotype cloning
WO2001081541A2 (en) * 2000-04-19 2001-11-01 Research Development Foundation Pcr-hybridization assays specific for integrated retroviruses

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992013101A1 (en) * 1991-01-25 1992-08-06 Ingeny B.V. Method of detecting dna sequence variation
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
WO2000022164A1 (fr) * 1998-10-15 2000-04-20 Genset Sondes fluorescentes de peinture chromosomique
WO2000024935A2 (en) * 1998-10-26 2000-05-04 Yale University Allele frequency differences method for phenotype cloning
WO2001081541A2 (en) * 2000-04-19 2001-11-01 Research Development Foundation Pcr-hybridization assays specific for integrated retroviruses

Also Published As

Publication number Publication date
CN1615367A (zh) 2005-05-11
CN1288252C (zh) 2006-12-06

Similar Documents

Publication Publication Date Title
US10697013B1 (en) Methods for analyzing nucleic acids from single cells
JP6571895B1 (ja) 核酸プローブ及びゲノム断片検出方法
JP6803327B2 (ja) 標的化されたシークエンシングからのデジタル測定値
JP3715657B2 (ja) 差引きハイブリダイゼーションおよび差異分析の方法
JP5916166B2 (ja) 組織試料中の核酸の局在化された検出、又は空間的検出のための方法及び生成物
JP3936798B2 (ja) Ｒｎａ標的配列の増幅方法
JP6441893B2 (ja) 標的配列の濃縮
TWI332987B (zh)
CN110719957B (zh) 用于核酸靶向富集的方法和试剂盒
JP2008508901A (ja) 核酸の単離および増幅方法
JP2004523201A5 (zh)
JP2015508995A (ja) 核酸ハイブリダイゼーションプローブ
WO2016165591A1 (zh) 基于焦磷酸测序技术的mgmt基因启动子甲基化检测
JP2023065620A (ja) Ｄｎａ配列のクラスター化のための方法
WO2020151283A1 (zh) 一种基于选择性消除野生链背景干扰的基因突变检测方法
JP4806150B2 (ja) 腫瘍細胞検出のための核酸プライマー及びプローブ
CN107083427B (zh) Dna连接酶介导的dna扩增技术
WO2011101744A2 (en) Region of interest extraction and normalization methods
WO2019090621A1 (zh) 钩状探针、核酸连接方法以及测序文库的构建方法
JP2024515305A (ja) 核酸の濃縮及び検出
WO2019023243A1 (en) METHODS AND COMPOSITIONS FOR SELECTING AND AMPLIFYING DNA TARGETS IN A SINGLE REACTION MIXTURE
US20230183789A1 (en) A method of detecting structural rearrangements in a genome
WO2004018691A1 (fr) Procede pour adn chromosomique micro-excise d'amplification et son utilisation
TWI811831B (zh) 用於檢測基因變異的靶向定序方法及套組
EP4012029B1 (en) Method for capturing nucleic acid molecule, preparation method for nucleic acid library, and a sequencing method

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CN US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 20028273087

Country of ref document: CN

122 Ep: pct application non-entry in european phase