CN101619335B - Method for preparing single chain DNA by using primer with stem-loop sample structure through PCR - Google Patents

Method for preparing single chain DNA by using primer with stem-loop sample structure through PCR Download PDF


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CN101619335B CN 200810132914 CN200810132914A CN101619335B CN 101619335 B CN101619335 B CN 101619335B CN 200810132914 CN200810132914 CN 200810132914 CN 200810132914 A CN200810132914 A CN 200810132914A CN 101619335 B CN101619335 B CN 101619335B
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stranded dna
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The invention belongs to the technical field of biology, and relates to a method for preparing single chain DNA by processes of preparing positive and negative chain products with different lengths, cutting the colloid and recycling by using primer with a stem-loop sample structure through PCR. In the invention, by downstream stem-loop sample structure primer designed regarding different DNA templates, PCR condition is improved, thus proving that PCR products are all expected two chains: a long chain and a short chain, and can be separated and purified easily through modified colloid. The invention has extensive application prospect in related single chain DNA preparation bio-medical research field comprising SELEX.


一种利用具有茎环样结构引物通过PCR制备单链DNA的方 For utilizing a square-like stem-loop structure by PCR using primers prepared single-stranded DNA


[0001] 发明领域: [0001] Field of the invention:

[0002] 本发明涉及一种利用具有茎环样结构引物通过PCR制备单链DNA的方法,主要特征是带有茎环结构的引物分子的使用,包括5'端的反向重复序列,GC含量以及二级结构; 涉及PCR反应条件;涉及发明中茎环结构的引物和PCR在单链DNA制备中的应用。 [0002] The present invention relates to a having a stem-loop-like structure primers by the method of single-stranded DNA prepared in the PCR, the main feature is the use of a primer molecule with stem-loop structure, comprising a 5 'inverted repeat sequence, the GC content of the terminal, and secondary structure; relates PCR conditions; primers and PCR applications involving stem-loop structure of the invention in the preparation of single-stranded DNA.

背景技术: Background technique:

[0003] 与RNA分子一样,单链DNA(ssDNA)结构灵活易变,三维结构复杂,很容易形成与靶标结合的口袋结构,因此单链DNA寡核苷酸配基(aptamer)在分子识别、化学分析及生物医药领域具有广阔应用前景。 [0003] As an RNA molecule, single-stranded DNA (ssDNA) flexible structure variable, complex three-dimensional structure, it is easy to form the pocket structure binding to a target, thus a single-stranded DNA oligonucleotide ligands (aptamers) identified in the molecule, chemical analysis and biomedical field has broad application prospects. 特别是在SELEX(Systematic Evolution of Ligandsby Exponential Enrichment)技术研究与应用中,ssDNA的制备更是关键。 Especially in SELEX (Systematic Evolution of Ligandsby Exponential Enrichment) technology research and application, is the key of ssDNA prepared. SELEX技术是通过反复将单链随机DNA文库与靶分子孵育、分离结合的单链DNA、PCR扩增,最终获得高亲和力的与靶分子特异结合的ssDNA 序列(aptamer) (Tuerk C, et al. Sciencel990 ;249 : 505-510)。 SELEX process is repeated by the random single-stranded DNA library is incubated with the target molecule, single-stranded DNA binding separation, the PCR amplification, ssDNA finally obtained sequence (aptamer) (Tuerk C, et al specifically binding to the target molecule with high affinity. Sciencel990; 249: 505-510). SELEX筛选的一个关键步骤是富集后单链DNA文库的再制备,将PCR产生的双链产物中的负链去除,保留具有结合能力的正链。 SELEX screening a key step is the preparation of single-stranded DNA library enriched again after the PCR-generated double stranded product of the negative strand is removed, to retain a positive strand having a binding ability. 假设某一ssDNA文库中混有dsDNA成分,那么随着筛选轮数的增加,文库中dsDNA的比例不仅会增加,而且能形成稳定的双螺旋结构不与靶分子结合,最终导致特异性的ssDNA配基丢失,SELEX筛选失败。 Suppose a ssDNA library mixed with dsDNA component, then as the number of rounds of selection, the library will not only increase the ratio of dsDNA, but also can form a stable double helix structure of the target molecule does not bind, resulting in specific ssDNA ligands the base is lost, SELEX screening failure. 除了SELEX筛选, 其它生物医学领域也常常需要制备单链的DNA。 SELEX screening addition, other biomedical applications often require the preparation of single stranded DNA. 因此,有效的ssDNA制备方法将便利生物医学研究。 Therefore, an effective preparation of ssDNA to facilitate biomedical research.

[0004] 当前,制备ssDNA的方法很多,如:直接化学合成、RNA逆转录、不对称PCR、亲和素标记磁珠分离等。 [0004] Currently, the method of preparing a lot of ssDNA, such as: direct chemical synthesis, RNA reverse transcription, the PCR asymmetric, magnetic bead separation avidin label and the like. 但在实际应用中,这些方法均存在一些问题,例如:1、直接化学合成法是方便简单,但是利用该法只能合成已知序列或事先设计好的随机序列ssDNA文库,对于经SELEX筛选富集获得的未知序列不适用。 However, in practice, these methods have problems, for example: 1, direct chemical synthesis method is a convenient and simple, but this method can only be synthesized by using a known pre-designed sequence or random sequence ssDNA library, screening for enriched by SELEX unknown sequence set obtained does not apply. 2、不对称PCR制备单链产物时产物容易发生弥散, 原因可能与PCR体系离子强度、dNTP浓度等有关,特别是以经过数轮SELEX筛选后的文库作为模板先PCR扩增成dsDNA,再经不对称PCR制备单链产物时更易出现弥散现象;另外,不对称PCR在前10-15个循环通常都产生双链产物,因此这种方法制备的单链纯度不高,存在互补负链干扰的弊端3、亲和素标记磁珠分离制备单链DNA时,生物素标记PCR产物的负链, 亲和素包被的磁珠分离的手段能获得高纯度的正链DNA,但该方法分离前需要先纯化PCR 产物去除生物素化引物的干扰,而且回收效率往往不能满足实验需求。 2, when preparing a single-stranded asymmetric PCR product is the product dispersion easily occurs, probably due to the ionic strength of PCR, dNTP concentration related systems, in particular in the library after several rounds of SELEX screening as a template for PCR amplification into first-dsDNA, and then by preparing single-stranded asymmetric PCR product was more susceptible to the phenomenon of diffusion; in addition, asymmetric PCR first 10-15 cycles typically produce double-stranded product, and therefore the purity of the single-stranded prepared by this method is not high, the presence of the complementary negative strand interference 3 prior drawbacks, when avidin labeled microbeads preparing single-stranded DNA, minus strand of the PCR product was labeled with biotin, avidin coated magnetic bead separation means plus strand DNA can be obtained with high purity, but the method of separating We need to remove the interference PCR product was purified biotinylated primer, and the recovery efficiency often can not meet the needs of the experiment. 磁珠价格昂贵也是制约实验的一个因素;另外,如果下游实验要用到生物素标记的正链DNA,则无法应用磁珠的方法制备单链;4、RNA逆转录制备单链DNA需要先将ssDNA转录成RNA,RNA分子容易降解造成配基的丢失,而且转录模板需要引入转录酶识别序列,这些序列如果和ssDNA退火, 便可能影响筛选,因此操作复杂不实用。 Magnetic beads is also a factor expensive experimental constraints; Further, if the downstream experiment to use biotin-labeled positive strand DNA, the beads can not be applied to a process for preparing a single-stranded; 4, RNA single-stranded DNA prepared by reverse transcription need to first ssDNA transcribed into RNA, RNA molecule susceptible to degradation caused by the loss of the ligand, the template for transcription and the need to introduce transcriptase recognition sequence, these sequences if ssDNA and annealed, it may affect the screening, a complicated operation and therefore impractical.

[0005] Kelly P. Williams (Nucleic acids research,1995,4220-4221)等介绍了一种新的利用特殊引物PCR制备单链的方法,该反义引物由三个部分组成,5'端加长序列-终止子-互补序列。 [0005] Kelly P. Williams (Nucleic acids research, 1995,4220-4221) and the like introduces a new method using PCR primers specific preparation of single strand, the antisense primer consists of three parts, the 5 'end of the longer sequence - terminator - complementary sequences. 互补序列与模板退火,发挥引物的功能;终止子为一个Taq酶不能穿越的非核苷酸物质,六乙烯乙二醇(HEGL)分子,用于阻止正义链的延伸;5'加长序列为20个dA组成的聚A尾巴,用于生成长链的PCR产物。 Complementary sequences anneal to a template, a primer function; terminator is a Taq enzyme can not pass through the non-nucleotide material, six ethylene glycol (HEGL) molecules, for preventing the sense strand extension; 5 'extended sequence of 20 dA consisting of a poly a tail, for generating PCR products of a long chain. 因此,反义引物和模板退火并在Taq酶作用下会延伸产生一个长于模板20个碱基的负链产物,而当正义引物以此反义链为模板延伸时,由于Taq酶不能越过HEGL,正链的延伸因此终止,故正链的长度与模板一样。 Accordingly, an antisense primer and template annealing and extends negative strand product is longer than the template 20 bases in the Taq enzyme, and when the sense primer in order antisense strand as a template extension, because Taq polymerase can not cross HEGL, Thus the normal chain terminating extension, so that the same length of the positive strand of the template. 在变性的PAGE 凝胶上,两条链可以明显分开进而分离纯化单链DNA。 On denaturing PAGE gel, two strands can be further isolated and purified clearly separated single-stranded DNA. 这种方法在单链DNA的制备方面具有明显的优越性,例如价格低廉、PCR —步获得等,但受修饰的限制,在合成引物时需要HEGL 修饰,很多公司不具备这种能力。 This method has the single-stranded DNA in the preparation of the obvious advantages, such as low price, PCR - obtaining step and the like, but modified by the limited modification required in the synthesis HEGL primers, many companies do not have this capability. 单链的加长序列也可能会和模板区退火影响扩增效率,尤其在体外筛选后期,随机区可能会含有多个dT等富集序列的情况时。 Long single-stranded sequences and may also affect the efficiency of amplification template annealing region, in particular in vitro screening late, when the random region may contain a plurality of sequences where dT enriched like.

[0006] 受上述思路启发,本发明巧妙地利用了二级结构的DNA模板阻止Taq酶延伸的特性,改进反义引物,不需要特殊修饰即可产生不同长度的PCR产物。 [0006] inspired by the above ideas, the present invention is the clever use of the DNA template secondary structure to prevent Taq enzyme extends characteristic, improved antisense primer, does not require special modification of PCR product can be of different lengths. 反义引物含有两部分,除了3'端的模板互补序列,5'端引进能形成茎环二级结构的一段反向重复序列,由于反向重复序列富含GC碱基,具有较高的Tm值,在Taq酶能较好的发挥作用的温度范围内(55°C-65°C)仍然会保持二级结构,从而阻止正链的延伸。 Antisense primer consists of two parts, in addition to a 3 'end complementary to the template sequence, 5' end of the introduction section capable of forming a stem-loop secondary structure of inverted repeats, inverted repeats because GC base-rich, has a high Tm value in the Taq enzyme can better functioning temperature range (55 ° C-65 ° C) still maintain the secondary structure, thereby preventing extension of the positive strand. 因此,PCR产物的两条链长度不同,相差反向重复序列的长度,在变性的PAGE凝胶上两条单链可以明显地分开进而分离纯化ssDNAo Thus, two different chain lengths of the PCR products, length difference between the inverted repeat, on a denaturing PAGE gel of two single strands may be separated clearly ssDNAo Further Purification

[0007] 本发明所述制备ssDNA的方法,除了可用于SELEX技术中,在基础医学、医药学中均可得到广泛应用。 The method of the invention is the preparation of ssDNA [0007] present, can be used in addition to the SELEX technology, the basic medicine, the medicine can be widely used.

发明内容: SUMMARY:

[0008] 本发明目的在于设计带有茎环结构的引物,利用PCR产生不同长度的正、负链产物,提出通过茎环结构引物和PCR制备单链DNA的方法。 [0008] The object of the present invention is to design primers having stem-loop structure, the use of different length PCR generated positive, negative strand product, made by primer stem structure and methods of preparation of single stranded DNA loop PCR.

[0009] 本发明通过以下技术方案实现: [0009] The present invention is achieved by the following technical solution:

[0010] 首先设计带有茎环结构的下游引物Pstemloop。 [0010] First, the downstream primer design with stem-loop structure Pstemloop. 其3'端为与模板互补的20个碱基,5'端为富含GC碱基的一段反向重复序列,能够产生一段长的互补茎和小的环结构,阻止Taq酶延伸,从而导致下游引物延伸产物长。 Its 3 'end is complementary to the template 20 bases 5' end is rich in GC base section of inverted repeat sequences, capable of producing a long stem and a complementary small ring structures, Taq enzyme extends prevented, resulting in the downstream primer extension product length. 上游引物为与模板互补的碱基序列。 An upstream primer complementary to the template nucleotide sequence.

[0011] PCR反应:变性程序与通常PCR同,退火条件根据引物序列互补区碱基序列而定, 延伸温度适当降低,设定在55°C _65°C之间,延伸时间适度延长,视模板长度而定。 [0011] PCR reactions: PCR with denaturation procedures and generally, annealing conditions according to the sequence complementary to the nucleotide sequence of the primer region may be extended due to lower temperature, is set between 55 ° C _65 ° C, extension time appropriately extended, depending on the template on the length.

[0012] PCR扩增后,通过变性胶鉴定两条产物链,切胶回收目的单链DNA。 [0012] Following PCR amplification, two product was identified by denaturing gel chains Gel Extraction target single-stranded DNA.

[0013] 本发明优点: [0013] The advantages of the present invention:

[0014] 1)本发明通过设计具有稳定茎环结构的引物和优化PCR反应条件,引物无需修饰,通过PCR反应即可获得不同长度的单链产物,直接切胶回收目的单链DNA。 [0014] 1) of the present invention has a stable stem-loop structure by designing primers and PCR conditions optimized, without modification of the primers, single-stranded product can be obtained by PCR of different lengths, directly Gel Extraction target single-stranded DNA. 因此,操作简单、省时、廉价,获得的单链DNA纯度高,回收效率高。 Thus, operation is simple, time saving, inexpensive, single-stranded DNA obtained in high purity, high recovery efficiency.

[0015] 2)本发明解决了传统SELEX操作中的单链DNA制备中的瓶颈问题,在其它涉及到单链DNA制备的生物医学领域也有广泛的应用价值,因而将有广阔的市场前景和经济效益。 [0015] 2) The present invention solves the bottleneck problem of preparing a single-stranded DNA conventional SELEX operation of, the other involving biomedical preparation of single-stranded DNA has wide applications, and therefore will have broad market prospect and economic benefit. 附图说明: BRIEF DESCRIPTION OF:

[0016] 图1本发明设计的引物产生的茎环结构。 [0016] FIG stem-loop structure of the invention is designed to produce a primer.

[0017] 图2茎环结构引物PCR扩增产生不同长度延伸产物原理图。 [0017] FIG. 2 stem-loop primers produce PCR amplification products of different length extending in the schematic. [0018] 图3不同模板单链DNA经茎环结构引物扩增产生不同长度单链产物的电泳图。 [0018] FIG 3 different template single stranded DNA stem-loop structure by electrophoresis primers of different lengths to produce single-stranded amplification product.

[0019] UGP45ssDNA 模板;2、GP45ssDNA 经莲环结构引物PCR 扩增的产物;3、LG45ssDNA 经茎环结构引物PCR扩增的产物;4、LG45ssDNA模板;M、双链DNA分子量标准。 [0019] UGP45ssDNA template; 2, GP45ssDNA ring structure by Lin primer PCR amplified product; 3, LG45ssDNA by the stem-loop structure primer PCR amplified product; 4, LG45ssDNA template; M, double-stranded DNA molecular weight standards.

[0020] 图4引物的延伸产物放射性自显影图。 [0020] 4 primer extension product FIG autoradiography FIG.

具体实施方式: detailed description:

[0021] 下面通过分别针对不同的单链DNA模板设计的茎环结构下游引物及PCR来详细说明本发明。 [0021] The present invention will be described in detail below by separately for different single-stranded DNA template design stem-loop structure and the downstream primer PCR.

[0022] 1.针对不同的单链DNA模板设计茎环结构的下游引物: [0022] 1 for different single-stranded template DNA stem-loop structure designed downstream primer:

[0023] GP45 模板(88nt): [0023] GP45 template (88nt):


[0025]引物: [0025] Primer:

[0026]上游引物(Plong-I) : 5,-GCAATGGTACGGTACTTCC-3, [0026] The upstream primer (Plong-I): 5, -GCAATGGTACGGTACTTCC-3,

[0027]下游引物(Pll) : 5,-TTAGCAAAGTAGCGTGCACTTTTG-3, [0027] The downstream primer (Pll): 5, -TTAGCAAAGTAGCGTGCACTTTTG-3,

[0028] 茎环结构下游引物(Pstemloop)(图1): [0028] The stem-loop structure downstream primer (Pstemloop) (FIG. 1):


[0030] LG45 模板(81nt): [0030] LG45 template (81nt):


[0032]引物: [0032] Primer:

[0033]上游引物(LGP5) :5,-GCCTGTTGTGAGCCTCCT [0033] The upstream primer (LGP5): 5, -GCCTGTTGTGAGCCTCCT

[0034] 茎环结构下游引物(LGP3stemloop)(图1): [0034] The stem-loop structure downstream primer (LGP3stemloop) (FIG. 1):


[0036] 注:下划线序列为含反向重复序列的茎环结构部分。 [0036] Note: The underlined sequence is inverted repeat stem-loop structure containing a partial sequence.

[0037] 上述模板及引物序列由^witrogen公司合成后,分别用双蒸水溶解,引物稀释成50 μ M,_20°C存放,备用。 [0037] The template and primer sequences synthesized by the company ^ witrogen were dissolved in double distilled water to dilute the primer 50 μ M, _20 ° C storage, backup.

[0038] 2.放射性同位素标记引物: [0038] 2. radioisotope labeled primer:

[0039] 利用T4 Polynucleotide Kinase和[γ-32P] ATP,通过磷酸化反应对合成的寡核苷酸引物的5,端进行放射性标记。 [0039] using T4 Polynucleotide Kinase and [γ-32P] ATP, by phosphorylation reactions to 5, the end of the synthetic oligonucleotide primer radiolabeled. 按DNA 5,末端标记试剂盒(MEGALABEL Kit, TaKaRaBiotechnology Co.,Ltd, D6070)说明进行。 Press DNA 5, end labeling kit (MEGALABEL Kit, TaKaRaBiotechnology Co., Ltd, D6070) instructions. 10 μ 1 体系中含有5-IOpmoIe 引物, 50 μ Ci [γ-32Ρ] ATP 及IOU Τ4ΡΝΚ。 10 μ 1 contained in the system 5-IOpmoIe primer, 50 μ Ci [γ-32Ρ] ATP and IOU Τ4ΡΝΚ. 37°C 磷酸化反应40min,70°C IOmin 灭活酶。 37 ° C phosphorylation 40min, 70 ° C IOmin inactivate the enzyme. 乙醇沉淀的方法去除大部分未反应的[Y-32P] ATP,纯化标记的引物。 To remove most of the ethanol precipitation method [Y-32P] ATP, purified labeled primer is unreacted.

[0040] 3. PCR 扩增: [0040] 3. PCR amplification:

[0041] PCR体系:100μ 1体系中含有10μ IlOX缓冲液,0. 2mM dNIPs,0. 5 μ M上游及下游引物,IOnM模板,2U Taq DNA聚合酶。 [0041] PCR system: 100μ 1 10μ IlOX system comprising a buffer, 0 2mM dNIPs, 0 5 μ M upstream and downstream primer, IOnM template, 2U Taq DNA polymerase...

[0042] PCR 条件:95°C预变性5min ;94°C变性lmin,37°C退火30sec,58°C延伸40sec,扩增30个循环;最后58°C终延伸anin。 [0042] PCR conditions: 95 ° C denaturation for 5min; 94 ° C denaturation lmin, 37 ° C annealing 30sec, 58 ° C extend 40sec, 30 cycles of amplification; extension end finally 58 ° C anin. [0043] 4.鉴定: [0043] 4. Identification:

[0044] 7M尿素10%变性聚丙烯酰胺凝胶电泳、Goldview染色或银染鉴定(图幻。或者放射性自显影分析引物延伸位置(图4)。[0045] 5.目的单链切胶回收: .. [0044] 7M urea denaturing 10% polyacrylamide gel electrophoresis, Goldview staining or silver staining identified (FIG phantom autoradiography analysis or primer extension position (FIG. 4) [0045] The object of the single-stranded Gel Extraction:

[0046] (1)将染色后的胶放在长波紫外灯上,打开紫外灯,观察DNA电泳条带; [0046] (1) A dyed glue on long wave ultraviolet light, the UV lamp is opened, DNA electrophoresis bands were observed;

[0047] (2)在防护罩防护下,锐利刀片切取目的条带,并移入1.5ml离心管中。 [0047] (2) In the protective shield, sharp blade cut the band of interest, and transferred to a 1.5ml microcentrifuge tube.

[0048] (3)加入400 μ 1 凝胶洗脱缓冲液(0. 5mol/L NH4Ac,0. 2% SDS, Im mol/L EDTA, pH 8.0, 7M尿素),于37°C洗脱6hr以上; [0048] (3) was added 400 μ 1 gel elution buffer (0. 5mol / L NH4Ac, 0. 2% SDS, Im mol / L EDTA, pH 8.0, 7M urea), eluting 6hr at 37 ° C the above;

[0049] (4)吸取洗脱液到新的1. 5ml离心管中,不要将凝胶吸出; [0049] (4) an eluent to draw new 1. 5ml centrifuge tube, the gel was not aspirated;

[0050] (5)加入1/100 体积的IM MgCl2,1/10 体积的3mol/LNaAc (ρΗ5· 2),2· 5 倍体积的无水乙醇,_70°C放置6hr以上; [0050] (5) was added 1/100 volume IM MgCl2,1 / 10 volumes of 3mol / LNaAc (ρΗ5 · 2), 2 · 5 volumes of absolute ethanol, _70 ° C 6hr disposed above;

[0051] (6)4°C,14000r/m,离心30min ; [0051] (6) 4 ° C, 14000r / m, centrifuged for 30 min;

[0052] (7)弃去上清,沉淀用4°C预冷的70%乙醇冲洗后离心同6 ; [0052] (7) The supernatant was discarded, the precipitate 4 ° C with 70% ethanol precooled centrifugation after washing with 6;

[0053] (8)弃去上清,沉淀于室温干燥; [0053] (8) The supernatant was discarded, the precipitate was dried at room temperature;

[0054] (9)将干燥好的DNA溶于适量三蒸水中。 [0054] (9) The dried DNA was dissolved in an appropriate amount of distilled water three. _20°C保存备用。 _20 ° C for use.

[0055] 实验结果: [0055] The results:

[0056] 利用茎环结构下游引物经PCR扩增产生了两条长度不等的正、负链产物,在变性胶上可明显得到分离。 [0056] The use of stem-loop structure downstream primer by PCR amplification produces two unequal length positive and negative strand product, on a denaturing gel to give significantly separated.

[0057] 由图3可以得到结论:茎环结构引物经PCR扩增产生了两条长度不等的正、负链产物,且该结构引物适用于不同的DNA模板。 [0057] FIG. 3 can be obtained from Conclusion: stem-loop structures by PCR amplification primers produced two unequal length positive and negative strand product, and the structure for different primer DNA template.

[0058] 由图4可以得出结论:茎环结构下游引物延伸产物为长链,较模板多出茎环结构部分的序列,因而迁移慢;上游引物延伸产物绝大部分与模板位置相同,为目的正链,少量由于下游引物茎环结构解链而产生长链。 [0058] FIG. 4 can be concluded: stem-loop structure downstream primer extension product is a long chain, compared with a template polynucleotide sequence portion of the stem-loop structure, and thus slow the migration; most of the same position with the template upstream primer extension product, is Objective normal chain, since a small amount of the downstream primer stem-loop structures and long-chain melting.

Claims (1)

  1. 1. 一种具有茎环结构的引物通过PCR制备单链DNA的方法,以单链DNA为模板,其特征在于,首先设计带有茎环结构的下游引物和不带茎环结构的上游引物,然后PCR扩增,扩增产物经过变性PAGE凝胶电泳,切胶回收目的单链DNA ;所述带有茎环结构的下游引物由3'端与单链DNA模板互补的20个碱基和5'端富含GC碱基的一段反向重复序列组成;所述PCR的延伸温度为55-65°C。 1. having a stem-loop structure of the primers by the method of single-stranded DNA prepared in the PCR, single-stranded DNA as a template, wherein the first design of the downstream primer with a stem-loop structure and the upstream primer without a stem-loop structure, then PCR amplification, amplification product was denatured PAGE gels, gel Extraction target single-stranded DNA; downstream primer having the stem loop structure composed of 3 end of single-stranded DNA complementary to the template base 20 'and 5 'GC-rich end of the base section of inverted repeat sequences; the PCR extension temperature of 55-65 ° C.
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