LoPPS: a long PCR product sequencing method for rapid characterisation of long amplicons

Here, we propose an optimised protocol (LoPPS, long PCR product sequencing) which allows the fast, cost-attractive, and high-throughput sequencing of long PCR products. LoPPS constitutes an alternative to the primer-walking technology which is expensive and time consuming but remains the current standard procedure. It is based on the ultrasonic shearing, polishing, and cloning of PCR or RT-PCR products and is compatible with 96- or 384-well microplate systems in which bacterial growth, preparation of plasmid DNA, and sequencing can be automated. We present results obtained from 24 different RT-PCR products (2.5-4.8 kbp long) obtained from various RNA viruses and fully sequenced using LoPPS. The method proved to be robust and fast. It was successfully used on a low amount of DNA and allowed each target nucleotide position to be controlled twice or more, with a final cost which is one-third of that of primer-walking.     

Sequencing of double-stranded PCR products

Very often the experimental step following PCR is sequencing of the amplified fragment. Two protocols that allow direct sequencing of a double-stranded PCR product are described. The first involves removal of one strand of the PCR product using an Ml3 single-stranded DNA clone, allowing the second strand to be sequenced. The second protocol involves Maxam-Gilbert chemical sequencing after PCR amplification with one labeled primer. The advantages and disadvantages of the two protocols are compared, but both yield DNA sequence without cloning of the PCR product.     

USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products

We present a method that allows simultaneous fusion and cloning of multiple PCR products in a rapid and efficient manner. The procedure is based on the use of PCR primers that contain a single deoxyuridine residue near their 5′ end. Treatment of the PCR products with a commercial deoxyuridine-excision reagent generates long 3′ overhangs designed to specifically complement each other. The combination of this principle with the improved USER cloning technique provides a simple, fast and very efficient method to simultaneously fuse and clone multiple PCR fragments into a vector of interest. Around 90% positive clones were obtained when three different PCR products were fused and cloned into a USER-compatible vector in a simple procedure that, apart from the single PCR amplification step and the bacterial transformation, took approximately one hour. We expect this method to replace overlapping PCR and the use of type IIS restriction enzymes in many of their applications.     

降落-重叠PCR法四重融合构建平菇同源重组片段

对多个长片段的基因融合目前仍缺少有效的方法. 本文提出一种新的融合PCR策略,即在常规的重叠PCR的第1步和第2步均增加1个降落PCR程序,减少不适当的退火温度和PCR产物3′端额外碱基A对片段融合、扩增的影响,提高正确融合与扩增的效率. 结果表明,为构建平菇葡聚糖合成酶启动子的同源重组序列,在4个长度分别是1 015 bp、2 822 bp、2 206 bp和1 008 bp的片段进行融合时,在重叠PCR的第1步加上退火温度61.5 ℃～57.5 ℃、每降落0.5 ℃进行1个循环的降落PCR程序,在重叠PCR的第2步加上退火温度60 ℃～56 ℃、每降落0.5 ℃进行1个循环的降落PCR程序,经过1次PCR即获得顺序正确的全长融合片段. 测序结果与4个片段序列的一致性达到98.5%,降落-重叠PCR法对多个长片段的基因 融合具有较高的应用价值.     

一种高效构建同源重组DNA片段的方法——融合PCR

融合PCR技术（fusion PCR）采用具有互补末端的引物,形成具有重叠链的PCR产物,通过PCR产物重叠链的延伸,从而将不同来源的任意DNA片段连接起来,此技术在不需要内切酶消化和连接酶处理的条件下实现DNA片段的体外连接,为同源重组片段的构建提供了快速简捷的途径。对原有的融合PCR技术进行改进,以三个同源重组线性DNA片段的构建为例,详细论述了改进的融合PCR技术的反应过程及技术体系。结果表明,改进的融合PCR技术可以同时进行三个片段及四个片段的融合反应,产物长度均在4.5kb以上,各同源重组片段在扩增过程中均无突变发生,获得的片段可以用于后续实验分析。     

Microsatellite analysis using a two-step procedure for fluorescence labeling of PCR products.

A method for fluorescent labeling of PCR products has been developed. This method consists in a two-step procedure in which a first exponential classical PCR is followed by a "linear amplification". This second step relies on incorporation of fluorescent dNTP (dUTP or dCTP) in order to label the product on only one strand. The products can be applied without prior purification directly to a gel on a fluorescence-based automated DNA sequencer, for length and allele determination. The reliability of the results equals those of the classical 32P or fluorescent primer labeling methods, and the method is definitely less costly. Since the interpretation of the results is easier than with the method consisting in a fluorescent dNTP uptake in both strands in a single PCR, the present strategy should prove useful in mapping projects requiring analysis of a large number of microsatellites.     

A novel method for producing partial restriction digestion of DNA fragments by PCR with 5-methyl-CTP.

Partial digestion of DNA fragments is a standard procedure for subcloning analysis and for generating restriction maps. We have developed a novel method to generate a partial digestion for any DNA fragment that can be amplified by PCR. The method involves the incorporation of 5-methyl-dCTP into the PCR product to protect most of the restriction sites. As a result, complete digestion of the modified PCR products with a 5-methyl-dCTP-sensitive enzyme will produce an array of restriction fragments equivalent to a partial restriction enzyme digestion reaction done on unmethylated PCR products. This method reduces the time and material needed to produce partially-digested DNA fragments by traditional methods. Furthermore, using fluorescein-labeled primers in the reaction, we were able to detect the fluorescein-labeled end fragments resulting from the enzyme digestion using a fluorimager or anti-fluorescein-AP antibody and thus determine the restriction maps.     

A simple method for direct automated sequencing of PCR fragments.

A simple and rapid method for direct sequencing of PCR-generated fragments has been developed for use on Applied Biosystems 373A Automated DNA Sequencer utilizing the DyeDeoxy terminator chemistry. Standard PCR conditions are used to generate a DNA fragment, which is subsequently gel-purified to remove excess primers and unwanted PCR products. The sequencing reactions are carried out in a thermal cycler using the purified product as template DNA and the Dye-Deoxy terminators. The sequence of 500-bp region in the bacteriophage lambda genome and a 320-bp fragment of the human genomic erythropoietin gene were sequenced with greater than 99% accuracy using this method.     

Whole genome sequence-enabled prediction of sequences performed for random PCR products of Escherichia coli

The sequence of an unknown PCR product generated by random (and conventional) PCR could be determined without sequencing when it is provided with the template DNA sequence. Theoretically, this was based on formerly established ideas which assert that the amount of random PCR product mainly depends on the stability of the primer-binding structures and that the dynamic solution structure of DNA is essentially governed by the Watson–Crick base pairing. However, it has not been clear whether this holds true for larger genomes of mega- to gigabase size, beside the λ phage genome (of 50 kb) used previously, nor has it been ascertained to uniquely specify the sequence of a random PCR product. Here, we jointly use two computer programs together with experimental data from Genome Profiling (i.e. TGGE analysis of random PCR products). The first procedure carried out by a newly remodeled computer program (PCRAna-A1) was shown to be competent to calculate a set of random PCR products from Escherichia coli genome DNA (4.7 Mb). The other procedure performed with another program (Poland-H) played a critical role in determining the final candidate sequence by theoretically offering the initial melting temperature and the melting pattern of unspecified candidate sequences. The success attained here not only proved our method to be useful for sequence prediction but also confirmed the above-mentioned ideas as rational. We believe that this is the first case to computer-utilize a genome sequence as a whole.     

Diagnosis of alpha 1-antitrypsin deficiency by enzymatic amplification of human genomic DNA and direct sequencing of polymerase chain reaction products.

We have compared sequencing of cloned "polymerase chain reaction" (PCR) products and the direct sequencing of PCR products in the examination of individuals from six families affected with alpha 1-antitrypsin (AAT) deficiency. In families where paternity was in question we confirmed consanguinity by DNA fingerprinting using a panel of locus-specific minisatellite probes. We demonstrate that direct sequencing of PCR amplification products is the method of choice for the absolutely specific diagnosis of AAT deficiency and can distinguish normals, heterozygotes and homozygotes in a single, rapid and facile assay. Furthermore, we demonstrate the reproducibility of the PCR and a rapid DNA isolation procedure. We have also shown that two loci can be simultaneously amplified and that the PCR product from each locus can be independently examined by direct DNA sequencing.     

A novel system for large-scale sequencing of cDNA by PCR amplification.

We have developed a method for constructing a library containing the 3' end fragment of cDNA for large-scale sequencing of cDNA clones. The average size of the insert was 270 bp. Cell lysates that carry plasmids having the cDNA insert were subjected to PCR amplification of the cDNA moiety and the products were subjected to sequencing analysis using an autosequencer. With this protocol, sample preparation became a non-limiting step, that allowed us to sequence as many samples as the autosequencer could handle.     

An efficient method for purification of PCR products for sequencing

A high-throughput DNA sequencing method that generated high quality data was developed. A frame fashioned from a standard agarose gel combined with 0.1%-0.2% low-melting point (LMP) agarose gel was used to isolate the PCR product of interest. Collected PCR products were centrifuged without any reagents and the supernatants were directly used for a sequencing reaction. This method is simple and labor efficient, provides high quality sequences at a low cost, and bypasses problems with impure PCR products. This technique has been used for single nucleotide polymorphism (SNP) discovery in Populus angustifolia trees.     

Fluorescent protein vector for directional selection of PCR clones

Green fluorescent protein (GFP) has become a convenient and versatile tool as a reporter protein in many aspects of science. Here, we show that the enhanced yellow fluorescent protein (EYFP) variant may be used advantageously as a reporter system for directional cloning of blunt-ended PCR products. We have constructed a pUC18-derived plasmid containing a reporter gene coding EYFP cloned into the BamHI/HindIII sites. The blunt-ended PCR product is cloned into the SmaI site of that plasmid. A reverse PCR primer must be designed with extra bases on the 5' end that are required to introduce a ribosome binding site (rbs) for EYFP expression. The reporter gene coding EYFP is not expressed unless an rbs is introduced in the proper orientation at the 3' end of the cloned PCR insert. The results of this cloning procedure may be analyzed by simple visual inspection using a transilluminator. In most cases, successful directional cloning results in white fluorescent colonies. The proposed procedure is a convenient method that can reduce the time- and labor-intensive analysis of the clones obtained during blunt-ended PCR product cloning.     

Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously

A procedure for precise assembly of linear DNA constructs as long as 20 kb is proposed. The method, which we call long multiple fusion, has been used to assemble up to four fragments simultaneously (for a 10.8 kb final product), offering an additional improvement on the combination of long PCR and overlap extension PCR. The method is based on Pfu polymerase mix, which has a proofreading activity. We successfully assembled (and confirmed by sequencing) seven different linear constructs ranging from 3 to 20 kb, including two 20 kb products (from fragments of 11, 1.7 and 7.5 kb), two 10.8 kb constructs, and two constructs of 6.1 and 6.2 kb, respectively. Accuracy of the PCR fusion is greater than or equal to one error per 6.6 kb, which is consistent with the expected error rate of the PCR mix. The method is expected to facilitate various kinds of complex genetic engineering projects that require precise in-frame assembly of multiple fragments, such as somatic cell knockout in human cells or creation of whole genomes of viruses for vaccine research.     

通过PCR进行长片段DNA的合成

利用PCR合成DNA长片段(Synthesis Large Frament DNA using PCR,SLFD PCR)是一种有效的合成长片段DNA的方法。采用一段已知的500～600bp碱基的DNA片段为PCR模板,根据所要合成的DNA序列可以设计一系列的PCR引物,这些引物都位于模板DNA的5’端,长度为50～60bp,且从5’到3’方向顺序重叠,重叠碱基数目为12～15,全部引物叠加所得到的DNA正是自己所要合成的DNA。这组引物中最3’端的一条含有一个BamH Ⅰ酶切位点,在该位点后面有15碱基与模板DNA5’端一致的序列。另外还设计一条与该模板匹配的下游引物,引物内也含有一个BamH Ⅰ酶切位点。首先采用5’端最右侧的引物与下游引物进行PCR,在PCR进行10个循环后,以此次PCR的产物为下一轮PCR的模板,该轮PCR采用右侧倒数第二个引物为上游引物,下游引物保持不变。采用类似的方法,完成所有的PCR循环,就可以得到所需要合成的DNA长片段。该方法尤其适合100～200碱基左右的长片段DNA的快速合成与克隆。     

Cloning and direct sequencing of plant promoters using primer-adapter mediated PCR on DNA coupled to a magnetic solid phase.

A method that allows amplification and direct sequencing or cloning of an unknown DNA segment flanked by a known sequence is described using barley genomic DNA. The method avoids the step of circularization necessary for inverse PCR by ligation of primer-adapters to restricted genomic DNA. Specificity is achieved in the first amplification step; linear PCR with a biotinylated primer complementary to the known flanking sequence (primer 1-B) produces a single-stranded product that is purified employing streptavidin-coated magnetic beads. After this step, which removes genomic DNA, two rounds of exponential PCR are performed, first with the adapter-primer and primer 1 and second with primer 1 substituted by a nested primer 2. If the second primer is biotinylated, the product can be sequenced directly using solid-phase sequencing. We have employed this method to sequence directly and to clone the promoters of two late embryogenesis-abundant (Lea) genes (B19.4 and B19.3) from barley. Lea B19.4 and B19.3 encode putative desiccation-protective proteins that act in the final stages of embryogenesis and have previously been cloned as cDNAs. We demonstrate here that their proximal promoter regions are very similar (80% identity) and that both contain putative abscisic acid-responsive elements.     

A-T linker adapter polymerase chain reaction for determining flanking sequences by rescuing inverse PCR or thermal asymmetric interlaced PCR products

The polymerase chain reaction (PCR)-based genome walking method has been extensively used to isolate unknown flanking sequences, whereas nonspecific products are always inevitable. To resolve these problems, we developed a new strategy to isolate the unknown flanking sequences by combining A-T linker adapter PCR with inverse PCR (I–PCR) or thermal asymmetric interlaced PCR (TAIL–PCR). The result showed that this method can be efficiently achieved with the flanking sequence from the Arabidopsis mutant and papain gene. Our study provides researchers with an additional method for determining genomic DNA flanking sequences to identify the target band from bulk of bands and to eliminate the cloning step for sequencing.     

Direct sequencing of PCR products using the Maxam-Gilbert method

Direct sequencing of polymerase chain reaction (PCR) products by using the Maxam-Gilbert method is described. In this method, one of the primers is end labeled. Thus it is possible to sequence the reaction product directly following purification using this chemical method.