Gc-rich Pcr Optimization
Gc-rich Pcr Optimization - Related Videos
12:49
Mutagenesis and Analysis of Genetic Mutations in the GC-rich KISS1 Receptor Sequence Identified in Humans with Reproductive Disorders
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Cited by 3 •
2011
Mutations in the kisspeptin receptor (KISS1R) are associated with reproductive disorders in patients. Here we describe how to introduce mutations of interest in the GC-rich sequence of KISS1R as well as the use of KISS1R constructs to characterize the degradation pathway of the receptor by immunoprecipitation and western blot.
06:01
Optimized PCR-based Detection of Mycoplasma
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Cited by 14 •
2011
The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.
09:22
Gas Chromatography (GC) with Flame-Ionization Detection
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2023
Source: Laboratory of Dr. B. Jill Venton - University of Virginia Gas chromatography (GC) is used to separate and detect small molecular weight compounds in the gas phase. The sample is either a gas or a liquid that is vaporized in the injection port. Typically, the compounds analyzed are less than 1,000 Da, because it is difficult to vaporize larger compounds. GC is popular for environmental monitoring and industrial applications because it is very reliable and can be run nearly continuously.
06:30
Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis
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Cited by 34 •
2014
PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.
07:27
High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs
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Cited by 13 •
2011
Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.
FAQs
Guanine and cytosine form strong base pairs, making GC-rich DNA more stable during amplification. This stability can hinder strand separation and encourage secondary structures, which may obstruct primer binding or polymerase movement. As a result, small changes in denaturation, annealing, or extension conditions can substantially affect amplification yield, specificity, and reproducibility.
Secondary structures within the template can fold regions of GC-rich DNA into shapes that limit access to primers or the polymerase. Even when the target sequence is present, these structures may prevent efficient copying or cause incomplete extension. Optimization aims to adjust reaction conditions so the polymerase can progress through difficult regions more consistently.
DMSO and betaine are additives used when standard amplification conditions do not adequately support a GC-rich target. They can be incorporated into optimization trials alongside adjusted thermal conditions and polymerase selection. Their purpose is to improve access to difficult template regions, helping increase the likelihood of specific, reproducible product formation.
An appropriate polymerase can improve amplification of templates that are difficult to copy because of high GC content and associated secondary structures. Polymerase choice should be evaluated together with denaturation, annealing, and extension settings rather than treated as an isolated variable. This combined optimization can improve product yield and the reliability of downstream analysis.
Begin by evaluating the denaturation, annealing, and extension conditions, since each stage can be affected by template stability or restricted polymerase progression. If amplification remains weak or inconsistent, test an additive such as DMSO or betaine and consider a different polymerase. Comparing these changes systematically helps identify conditions that improve specificity and yield.
Reliable amplification supports gene analysis, mutation detection, sequencing, cloning, and characterization of GC-rich genomic regions. In these applications, improved yield and specificity help ensure that the target can be examined or used in subsequent experimental steps. Reproducible amplification is particularly important when a difficult region must be compared across samples or processed further.