阳光下的日耳曼尼亚 -
Qβ复制酶反应
Kacian等于1972年首次报报Qβ复制酶催化RNA模板的自我复制功能，它能在常温30min，将其天然MDV扩增至109.1986年Chu等报道用生物标记的靶序列特异性探针，可与亲和素联接的MDV杂交，经洗脱未被结合的MDV后，再加入Qβ复制酶，扩增复制MDV拷贝，然后用溴乙锭染色检测或用同源性的第二探针杂交.
Qβ复制酶是一种RNA指导的RNA聚合酶，它有3个特点:①不需寡核苷酸引物的引导就可启动RNA的合成.②能特异地识别RNA基因中由于分子内碱基配对而形成的特有的RNA折叠结构.③在Qβ复制酶的天然模板MDV的非折叠结构区插入一短的核酸序列不影响该酶的复制.因而，如在此区插入核酸探针，则其序列照样可能被Qβ复制酶扩增.
1988年Lizardi等，将靶基因序列插进MDV质粒里，用T7RNA聚合酶催化转录出MDV探针，这种RNA探针可与靶序列杂交，然后洗去非杂交的探针，加入Qβ复制酶来扩增探针，被扩增的探针又可作为模板进行扩增，并呈指数递增.其产物按上述两种方法进行检测.现在该技术又发展了夹心杂交法，分子开关和靶依赖的复制等技术.
其扩增状况，此法可用来检测基因的突变，染色体重排或转位，基因缺失及微生物的型别鉴定等.
反向PCR
反向PCR是用反向的互补引物来扩增两引物以外的未知序列的片段，而常规PCR扩增的是已知序列的两引物之间DNA片段.实验时选择已知序列内部没有切点的限制性内切酶对该段DNA进行酶切，然后用连接酶使带有粘性末端的靶序列环化连接，再用一对反向的引物进行PCR，其扩增产物将含有两引物外未知序列，从而对未知序进行分析研究.
不对称PCR
不对称PCR是用不等量的一对引物，PCR扩增后产生大量的单链DNA这对引物分别称为非限制引物与限制性引物，其比例一般为50～100∶1.在PCR反应的最初10～15个循环中，其扩增产物主要是双链DNA，但当限制性引物低浓度引物消耗完后，非限制性引物高浓度引物引导的PCR就会产生大量的单链DNA.不对称PCR的关键是控制限制性引物的绝对量，需多次摸索优化两条引物的比例.还有一种方法是先用等浓度的引物PCR扩增，制备双键DNA，然后以此dsDNA为模板，再以其中的一条引物进行第二次PCR，制备ssDNA.不对称PCR制备的ssDNA，主要用于核酸序列测定.
重组PCR
使两个不相邻的DNA片段重组在一起的PCR称为重组PCR，1986年报道了由PCR扩增的两个DNA片段通过重组合后再经延伸而制备出新的DNA分子.其基本原理为将突变碱基，插入或缺失片段，或一种物质的几个基因片段均设计在引物中，先分段对模板扩增，除去多余的引物后，将产物混合，再用一对引物对其进行PCR扩增.其产物将是一重组合的DNA.重组PCR主要用于位点专一碱基置换，DNA片段的插入或缺失DNA片段的连接(如基因工程抗体
多重PCR
一般PCR仅应用一对引物，通过PCR扩增产生一个核酸片段，主要用于单一致病因子等的鉴定.多重PCR，又称多重引物PCR或复合PCR，它是在同一PCR反应体系里加上二对以上引物，同时扩增出多个核酸片段的PCR反应，其反应原理，反应试剂和操作过程与一般PCR相同.
免疫PCR
免疫试验的主要步骤有三个:①抗原抗体反应，②与嵌合连接分子结合，③PCR扩增嵌合连接分子中的DNA一般为质粒DNA该技术的关键环节是嵌合连接分子的制备.在免疫-PCR中，嵌合连接分子起着桥梁作用，它有两个结合位点，一个与抗原抗体复合物中的抗体结合，一个与质粒DNA结合，其基本原理与ELISA和免疫酶染色相似，不同之处在于其中的标记物不是酶而是质粒DNA，在操作反应中形成抗原抗体-连接分子复合物，通过PCR扩增DNA来判断是否存在特异性抗原.
免疫PCR优点为:①特异性较强，因为它建立在抗原抗体特异性反应的基础上.②敏感度高，PCR具有惊人的扩增能力，免疫PCR比ELISA敏感度高105倍以上，可用于单个抗原的检测.③操作简便，PCR扩增质粒DNA比扩增靶基因容易得多，一般实验室均能进行.
☆ PCR用于进化分析
进化遗传学具有两个并列的研究方向：系统发育的重建和种群分析。自1962年， Zuckerkandl和Pauling提出蛋白质序列和基因序列的比较可以象分子种一样用于标志现存物种分化的时间以来，各种生化方法被用于系统发育的研究。在最初二十年内，同功酶的电泳分析、免疫学比较和蛋白质序列分析被广泛地应用。而最近，DNA杂交和核糖体RNA序列分析为分类学做出了重要贡献。这些技术大多有局限性，因为它们是估计而不是直接测量序列的差别。
☆ 反向聚合酶链反应
通常测定一个与已知序列相邻的DNA序列是必要的，例如位于编码DNA的上游和下游两 侧的区域，转位因子的插入位点以及克隆于Lambda、科期粒或酵母人工染色体载体上 的DNA片段末段的未知序列的探针等。这种末端特异探针在Southern Blot或染色体要得到边侧序列的探针一般需要进行一系列费时、费力的工作，首先用内切酶裂解和 用已知边侧序列的探针Southern杂交以确定大小适合于克隆的末端片段;这些片段还 要经过凝胶分离、克隆，得到的物质再与已知边侧区域杂交以确定合适的克隆子。要 测定未吞边侧区序列时，通常需要从克隆中进行各种片段的亚克隆。
为避免这些步骤，我们采用扩展的PCR方法，使相邻边侧区域得以扩增。典型的PCR扩增使用与互补链杂交的寡聚核苷酸引物。引物是定向的，使延伸向内跨过两个引物之间的区域。一个引物的DNA合成产物作为另一个引物的模板，进行DNA变性、引物退 火，DNA聚合酶管伸反应的多次重复性循环，可使引物规定区域的拷贝数成指数增 加。但用传统PCR方法得不到紧邻引物外侧的DNA序列，因为寡聚核苷酸所引导的既有 目的DNA又有引物外侧区的DNA合成在拷贝过程中只呈线性增长，这种线性增长是因 为，对于每种引物来讲，其不能引导DNA反向合成几乎是同时，有三个实验室分别设计出一种方法，使PCR可以扩增边侧区域。该方法反向PCR的基本点是用适当内切酶裂解核心区外分子，使这些酶切片段自身连接形成环状分子，从而将边侧区域转化为内部区域。
反向PCR程序
用传统的缓冲液和其他提供者推荐的条件裂解DNA。反向PCR所扩增的片段的大小由 PCR扩增片段的大小决定，目前，PCR扩增的实际上限为3kb。在许多情况下，首先 需要进行Southern杂交来确定内切酶用以产生大小适于环化及反向PCR的片段的末端片段。能裂解核心区的内切酶使反向PCR只能扩增引物所定模板依赖于引物的上游或上游区，而不裂解核心区的酶则使两上边侧序列都扩增，并带有由内切酶和环化类 型决定的接点例如，互补突头连接与钝头连接。对于扩增左翼或右翼序列，初试时 最好靠近识别上个碱基位位的酶，并已知在核心区有其方便的裂解位点。如果用反向 PCR从含有大量不同的克隆片段的同一载体中探测杂交探针，建议事先在载体中引入 合适的酶切位点。
用T4连接酶在稀DNA浓度下环化更容易形成单环。在一些实验中，为产生对反向PCR大小适当的DNA片段需要两种内切酶，但这样所产生的片段末端则不适于连接，环 化前需用Klenow或噬菌体T4DNA聚合酶修理钝化。连接前，需用酚或热变性使内切 酶失活。在我们实验中，不必裂解环状分子核心区也可得到有效的PCR扩增。这显然不同于Silver和Keerikatter[7]的实验结果，他们报道在核心裂解使模板线性化后，PCR扩增率增加100倍，但Triglia等则发现裂解环状分子与加热引起随机缺口效果相同。
反向PCR的应用
反向PCR的应用已经证明该方法可以避免不方便的克隆和亚克隆步骤，因此可解决大 量问题。我们最初用反向PCR扩增E.coli天然分离物中转位插入序 列ISL的边侧序列;Triglia等将反向PCR用于编码疟原虫主裂殖子表面抗原前体的基因，在实验中，他们用RsaⅠ酶裂解基因组DNA，连接，得到的环再用HinfⅠ在内部位点酶切，然后进行扩增，得到预期的297bp大小的片段，并用DNA直接测序进行鉴定。他们认为反向PCR由于具有从全长cDNA得到序列信息的优 点，将对步查现转录基因的5端或3端的边侧区域有用。
反向PCR的另一个应用是Silver和Keerikatte进行的。他们将其应用于扩增拉于整合在小鼠细胞中的外生原病毒DNA边侧的细胞DNA。除强调反向PCR在染色体“步查” 或“跳查”中的用途，他们还指出，该技术用于扩增特征性弱的序列，这 些序列在E.Coli或其他宿主载体系统中很骓或不能克隆。
康康map -
* regular basic PCR
* Allele-specific PCR: a diagnostic or cloning technique which is based on single-nucleotide polymorphisms (SNPs) (single-base differences in DNA). It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3" ends encompass the SNP. PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence. See SNP genotyping for more information.
* Assembly PCR or Polymerase Cycling Assembly (PCA): artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments, thereby selectively producing the final long DNA product.
* Asymmetric PCR: preferentially amplifies one DNA strand in a double-stranded DNA template. It is used in sequencing and hybridization probing where amplification of only one of the two complementary strands is required. PCR is carried out as usual, but with a great excess of the primer for the strand targeted for amplification. Because of the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required. A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.
* Helicase-dependent amplification: similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles. DNA helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.
* Hot-start PCR: a technique that reduces non-specific amplification during the initial set up stages of the PCR. It may be performed manually by heating the reaction components to the melting temperature (e.g., 95°C) before adding the polymerase. Specialized enzyme systems have been developed that inhibit the polymerase"s activity at ambient temperature, either by the binding of an antibody[9] or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.
* Intersequence-specific PCR (ISSR): a PCR method for DNA fingerprinting that amplifies regions between simple sequence repeats to produce a unique fingerprint of amplified fragment lengths.
* Inverse PCR: is commonly used to identify the flanking sequences around genomic inserts. It involves a series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown sequence.
* Ligation-mediated PCR: uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers; it has been used for DNA sequencing, genome walking, and DNA footprinting.
* Methylation-specific PCR (MSP): developed by Stephen Baylin and Jim Herman at the Johns Hopkins School of Medicine, and is used to detect methylation of CpG islands in genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCRs are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation.
* Miniprimer PCR: uses a thermostable polymerase (S-Tbr) that can extend from short primers ("smalligos") as short as 9 or 10 nucleotides. This method permits PCR targeting to smaller primer binding regions, and is used to amplify conserved DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene.
* Multiplex Ligation-dependent Probe Amplification (MLPA): permits multiple targets to be amplified with only a single primer pair, thus avoiding the resolution limitations of multiplex PCR (see below).
* Multiplex-PCR: consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis.
* Nested PCR: increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3" of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.
* Overlap-extension PCR: a genetic engineering technique allowing the construction of a DNA sequence with an alteration inserted beyond the limit of the longest practical primer length.
* Quantitative PCR (Q-PCR): used to measure the quantity of a PCR product (commonly in real-time). It quantitatively measures starting amounts of DNA, cDNA or RNA. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. Quantitative real-time PCR has a very high degree of precision. QRT-PCR methods use fluorescent dyes, such as Sybr Green, EvaGreen or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time. It is also sometimes abbreviated to RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions, since RT-PCR commonly refers to reverse transcription PCR (see below), often used in conjunction with Q-PCR.
* Reverse Transcription PCR (RT-PCR): for amplifying DNA from RNA. Reverse transcriptase reverse transcribes RNA into cDNA, which is then amplified by PCR. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites. If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of exons and introns in the gene. The 5" end of a gene (corresponding to the transcription start site) is typically identified by RACE-PCR (Rapid Amplification of cDNA Ends).
* Solid Phase PCR: encompasses multiple meanings, including Polony Amplification (where PCR colonies are derived in a gel matrix, for example), Bridge PCR (primers are covalently linked to a solid-support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR (where conventional Solid Phase PCR can be improved by employing high Tm and nested solid support primer with optional application of a thermal "step" to favour solid support priming).
* Thermal asymmetric interlaced PCR (TAIL-PCR): for isolation of an unknown sequence flanking a known sequence. Within the known sequence, TAIL-PCR uses a nested pair of primers with differing annealing temperatures; a degenerate primer is used to amplify in the other direction from the unknown sequence.
* Touchdown PCR (Step-down PCR): a variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3-5°C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3-5°C) below the primer Tm. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles.
* PAN-AC: uses isothermal conditions for amplification, and may be used in living cells.
* Universal Fast Walking: for genome walking and genetic fingerprinting using a more specific "two-sided" PCR than conventional "one-sided" approaches (using only one gene-specific primer and one general primer - which can lead to artefactual "noise")[34] by virtue of a mechanism involving lariat structure formation. Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested PCR for rapid amplification of genomic DNA ends), 5"RACE LaNe and 3"RACE LaNe.
阿里阿涅德 -
PCR种类太多了
举几种常见的PCR吧：
荧光定量PCR，TAIL-PCR，重组PCR，降落PCR