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Amplification-Free Library Preparation for Paired-End Illumina Sequencing

The library preparation step is of critical importance for the quality of next-generation sequencing data. The use of the polymerase chain reaction (PCR) as a part of the standard Illumina library preparation protocol causes an appreciable proportion of the obtained sequences to be dupl ...

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Preparation of Next-Generation Sequencing Libraries Using Nextera Technology: Simultaneous DNA Fragmentation and Adaptor Tagging by In Vitro Transposi

DNA library preparation is a common entry point and bottleneck for next-generation sequencing. Current methods generally consist of distinct steps that often involve significant sample loss and hands-on time: DNA fragmentation, end-polishing, and adaptor-ligation. In vitro tr ...

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Determining DNA Methylation Profiles Using Sequencing

Cytosine methylation is an epigenetic mark that has a significant impact on the regulation of transcription and replication of DNA. DNA methylation patterns are highly conserved across cell divisions and are therefore highly heritable. Furthermore, in multicellular organisms, D ...

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The Use of the Polymerase Chain Reaction and the Detection of Amplified Products

The most polymorphic genetic markers are DNA regions composed of variable number tandem repeats (VNTRs) (1,2). Detection of the various VNTRs is possible by restriction fragment-length polymorphism (RFLP) analysis using the Southern blot procedure (3). However, this procedure is ti ...

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Mapping MHC Class II Genes and Disease-Susceptibility: Use of Polymerase Chain Reaction and Dot Hybridization for Human Leukocyte Antigen Allele Typin

The genes of the human leukocyte antigen (HLA) region control a variety of functions involved in the immune response and influence susceptibility to over 40 diseases. The region maps to the short arm of chromosome 6 and is divided into three regions, denoted class I, II, and III. The HLA class II gene complex ...

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The Use of Polymerase Chain Reaction for Chromosome Assignment

The polymerase chain reaction (PCR) approach to chromosome assignment consists of analyzing the profile of specific amplification products of the gene of interest using genomic DNAs from a panel of somatic cell hybrids as templates (see Fig 1). Each hybrid somatic cell line contains the nor ...

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Polymerase Chain Reaction Analysis of DNA from Paraffin-Embedded Tissue

One of the greatest potentials of polymerase chain reaction (PCR) lies in the fact that even minute amounts of target DNA or extensively damaged DNA can be successfully amplified in vitro and thus become amenable to further study. This enables a detailed molecular analysis of small amounts of DNA f ...

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Use of Polymerase Chain Reaction for Screening Transgenic Mice

The production of transgenic mice by the direct microinjection of DNA fragments into isolated mouse embryos is now a standard technique for molecular and developmental analysis (1). Traditionally, the initial screening of litters of mice for those carrying the transgene has been carri ...

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Quantitation and Purification of Polymerase Chain Reaction Products by High-Performance Liquid Chromatography

The polymerase chain reaction (PCR) has rapidly become a standard laboratory technique. With the continuous development of PCR technology there is now a growing need for PCR product quantitation in areas such as therapeutic monitoring and quality control, disease diagnosis, and regul ...

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Nonradioactive Labeling of Polymerase Chain Reaction Products

Polymerase chain reaction (PCR) was originally introduced to amplify in vitro particular DNA sequences by the application of temperature cycles (1). In a modification, RNA molecules also may serve as templates by an additional reverse transcription step converting RNA in complement ...

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Use of Arithmetic Polymerase Chain Reaction for Synthesis of Single-Stranded Probes for S1 Nuclease Assays

The S1 nuclease protection assay involves the hybridization in solution of a single-stranded DNA probe to RNA, followed by digestion with S1 nuclease, which is specific for single-stranded nucleic acid (1). The protected probe is measured by first resolving the sample by denaturing gel elec ...

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Anchoring a Defined Sequence to the 5 Ends of mRNAs: The Bolt to Clone Rare Full Length mRNAs and Generate cDNA Libraries from a Few Cells

Among numerous applications, the polymerase chain reaction (PCR) (1,2) provides a convenient means to clone 5′ ends of rare mRNAs and to generate cDNA libraries from tissue available in amounts too low to be processed by conventional methods. Basically, the amplification of cDNAs by the PCR req ...

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Amplification of Gene Ends from Gene Libraries by Polymerase Chain Reaction with Single-Sided Specificity

Isolation of a full-length gene on the basis of a limited sequence information is often troublesome and challenging. Tremendous effort is needed to isolate a specific gene by screening cDNA or genomic libraries by oligonucleotide or nucleic acid probes. In those methods, basically nucleic ...

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cDNA Cloning by Inverse Polymerase Chain Reaction

Since the first report on cDNA cloning in 1972 (1), this technology has been developed into a powerful and universal tool in the isolation, characterization, and analysis of both eukaryotic and prokaryotic genes. But the conventional methods of cDNA cloning require much effort to generate a li ...

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Single Specific Primer-Polymerase Chain Reaction (SSP-PCR) and Genome Walking

The polymerase chain reaction (PCR) is used for selective amplification of DNA fragments from both prokaryotes and eukaryotes (1–3). The only requirement for amplification is that the sequence of the extremities of the DNA fragment to be amplified be known (4). This places a limitation on the use of ...

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Use of Degenerate Oligonucleotide Primers and the Polymerase Chain Reaction to Clone Gene Family Members

As more and more genes are cloned and sequenced, it is apparent that nearly all genes are related to other genes. Similar genes are grouped into families. Examples of gene families include the collagen, globin, and myosin gene families. There are also gene superfamilies. Gene superfamilies are co ...

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PCR-Based Full-Length cDNA Cloning Utilizing the Universal-Adaptor/Specific DOS Primer-Pair Strategy

Biological systems are often influenced by molecules that are neither present in vast quantities or easily purified to homogeneity from other cellular constituents. The development of simple, efficient molecular cloning systems coupled with the relative ease of DNA sequence dete ...

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Selection of Primers for Polymerase Chain Reaction

One of the most important factors affecting the quality of polymerase chain reaction (PCR) is the choice of primers. Several rules should be observed when designing primers and, in general, the more DNA sequence information available, the better the chance of finding an “ideal” primer pair. For ...

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Generation of a Polymerase Chain Reaction Renewable Source of Subtractive cDNA

Differential (+/-) first-strand cDNA screening methods identify clones corresponding to mRNAs that are expressed at a higher level in one of a pair of phenotypically different cells. This approach is limited by the fact that screening of libraries with labeled first-strand cDNAs synthes ...

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Site-Directed Mutagenesis by Double Polymerase Chain Reaction: Megaprimer Method

The “megaprimer” method (1) based on polymerase chain reaction (PCR) is one of the simplest and most versatile procedures of site-specific in vitro mutagenesis available to date. The method utilizes three oligonucleotide primers and two rounds of PCR performed on a DNA template containing ...

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