Technology
Nucleic Acid Extraction Guide: Methods, Purity, Yield & PCR Compatibility
Compare magnetic bead, column-based, manual and automated extraction methods for different sample types and downstream PCR workflows.
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Compare magnetic bead, column-based, manual and automated extraction methods for different sample types and downstream PCR workflows.
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A technique designed to rapidly enrich and amplify targets derived from samples that yield low levels of nucleic acids, such as formalin-fixed paraffin-embedded (FFPE) tissues, for use in next-generation sequencing. Briefly, total nucleic acid or genomic DNA (gDNA) is isolated from fresh or FFPE tissues. Total nucleic acid is converted to double-stranded cDNA (dscDNA) and cleaned by solid-phase reversible immobilization (SPRI). Then the dscDNA or gDNA is subjected to end repair, adenylation and ligation with a universal half-functional adapter. The modified DNA sample is then subjected to two rounds of nested low-cycle PCR, where the first round uses target gene-specific primers and the second round uses nested target gene-specific primers that are 5' tagged with a common sequencing adapter. The resulting target amplicon library is cleaned using SPRI and is suitable for use in next generation genomic assays.
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A molecular genetic technique that isolates and sequences chromosomal regions that are rich in open chromatin. First, nuclei are harvested from a cellular sample. Then a hyperactive Tn5 transposase is added to the nuclei where it excises non-nucleosomal DNA strands and ligates co-administered high-throughput sequencing adapters (tagmentation). The tagged DNA fragments are isolated, amplified by PCR and sequenced. The number of reads for specific region of DNA correlate with increased chromatin accessibility and this method can identify regions of transcription factor and nucleosome binding.
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An electrophoresis procedure, in which a sample is introduced into a capillary tube and the components are separated by the application of high voltage. Due to the large surface to volume ratio, cooling is more efficient, allowing use of higher voltages. As a result, separation occurs more quickly and sensitivity is increased. The technique allows for separation of proteins, nucleic acids, and carbohydrates.
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Any method that extracts and purifies DNA from biological samples, which can include the proper handling of biological samples prior to nucleic acid isolation, the separation of DNA from the rest of the components in the samples, and the storage of the DNA isolates.
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Gel electrophoresis is a method of separating large molecules (such as DNA fragments or proteins) from a mixture of similar molecules by electrophoretic filtration through a gelatinous matrix. Subject to an electric field, each molecule in the sample mixture migrates through the matrix at a different rate, depending mainly on electrical charge and size, resulting in spatial separation within the matrix of each molecular species in the sample mixture. Agarose and acrylamide in a cylindrical tube or slab gel are commonly used media for electrophoresis of proteins and nucleic acids.
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A type of nucleic acid amplification test in which sample DNA is exposed to a single temperature in the presence of multiple sets of primers and a polymerase with replication and high strand displacement activity. This test negates the need for thermal cyclers and may be a low cost alternative to PCR.
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Analytical and separative procedures in which molecules, particularly proteins or nucleic acids, are separated by their different electrophoretic mobilities in a hydrated gel. The gel suppresses convective mixing of the fluid phase through which the electrophoresis takes place and contributes molecular sieving. For proteins, commonly carried out in the presence of the anionic detergent sodium dodecylsulphate (SDS). SDS denatures proteins so that noncovalently associating sub unit polypeptides migrate independently and by binding to the proteins confers a net negative charge roughly proportional to the chain weight.
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A procedure for amplification of nucleic acid sequences from a messenger RNA template between a defined internal site and unknown sequences at either the 3'- or 5'-end of the mRNA.
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Match sample types with extraction, assay and workflow considerations for practical PCR planning.
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An isothermal, in vitro nucleic acid amplification method that allows primer-directed amplification of a target sequence. It utilizes a primer that can be nicked to create a polymerase start site and a DNA polymerase that will displace a downstream strand as it tracks along the template.
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A technique used to selectively replicate nucleic acid sequences from RNA templates using reverse transcriptase and RNA polymerase. Reverse transcriptase generates cDNA from the region of interest using primers and then RNA polymerase generates copies of the original RNA template.