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SYBR Green fluorescence chart produced in real-time PCR Melting curve produced at the end of real-time PCR. A real-time polymerase chain reaction (real-time PCR, or qPCR when used quantitatively) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR).
Since the late 1990s product analysis via SYBR Green, other double-strand specific dyes, or probe-based melting curve analysis has become nearly ubiquitous. The probe-based technique is sensitive enough to detect single-nucleotide polymorphisms (SNP) and can distinguish between homozygous wildtype , heterozygous and homozygous mutant alleles by ...
Due to the sensitivity of high-resolution melting analysis, it is necessary to carefully consider PCR cycling conditions, template DNA quality, and melting curve parameters. [12] For accurate and repeatable results, PCR thermal cycling conditions must be optimized to ensure that the desired DNA region is amplified with high specificity and ...
SYBR Green I (SG) is an asymmetrical cyanine dye [1] used as a nucleic acid stain in molecular biology. The SYBR family of dyes is produced by Molecular Probes Inc., now owned by Thermo Fisher Scientific. SYBR Green I binds to DNA. The resulting DNA-dye-complex best absorbs 497 nanometer blue light (λ max = 497 nm) and emits green light (λ ...
A master mix is a mixture containing precursors and enzymes used as an ingredient in polymerase chain reaction techniques in molecular biology. Such mixtures contain a mixture dNTPs (required as a substrate for the building of new DNA strands), MgCl 2, Taq polymerase (an enzyme required to building new DNA strands), a pH buffer and come mixed in nuclease-free water.
SYBR Green When the SYBR Green binds to the double-stranded DNA of the PCR products, it will emit light upon excitation. The intensity of the fluorescence increases as the PCR products accumulate. This technique is easy to use since designing of probes is not necessary given lack of specificity of its binding.
Polymerase cycling assembly (or PCA, also known as Assembly PCR) is a method for the assembly of large DNA oligonucleotides from shorter fragments. The process uses the same technology as PCR, but takes advantage of DNA hybridization and annealing as well as DNA polymerase to amplify a complete sequence of DNA in a precise order based on the single stranded oligonucleotides used in the process.
qPCR is unable to distinguish differences in gene expression or copy number variations that are smaller than twofold. On the other hand, dPCR has a higher precision and has been shown to detect differences of less than 30% in gene expression, distinguish between copy number variations that differ by only 1 copy, and identify alleles that occur ...