PCR Techniques

This video demonstrates the role of digital droplet polymerase chain reaction in identifying indel mutation in a DNA sample. The high precision and sensitivity of ddPCR enable accurate and consistent detection of rare mutations present in a sample, even at low levels. It can identify and quantify indel mutations in different regions of the genome, providing valuable insights into the mechanisms of mutation and the functional consequences of these mutations.

This video demonstrates the gradient polymerase chain reaction for optimization of the annealing temperature of primers, which sets gradient annealing temperature along wells. The effect of different annealing temperatures is determined by the yield of amplified products that helps to optimize the reaction condition.

In this video, we demonstrate the duplex digital PCR (ddPCR) technique — a modification of the traditional PCR technique that is useful in detecting two different genetic markers simultaneously. A single PCR reaction is partitioned into nanoliter-sized emulsified droplets that are independently amplified, and the detection of differently colored fluorescence amplification signals from the fraction of droplets is used to compute the initial concentration of the target sequences.

This video demonstrates a quantitative method for detecting viral infection using real-time reverse transcription PCR. The reverse transcription step converts the RNA to cDNA, which is then amplified via PCR cycles. The presence of viral RNA in the sample is confirmed by analyzing the amplification and dissociation curves of DNA obtained from the PCR.

This video describes wild-type blocking polymerase chain reaction, or WTB-PCR, which detects somatic mutations. The PCR-based technique uses a locked nucleic acid, or LNA, an oligonucleotide that binds to its complementary wild-type allele and blocks its elongation. The blocking selectively allows the amplification of low-level mutant alleles over their wild-type variants in the sample.

In this video, we discuss the quantitative polymerase chain reaction, qPCR, to enumerate T7 bacteriophages through DNA quantification. The fluorescent dye in the PCR mixture binds with the newly synthesized DNA strands and emits fluorescence, which is measured.

This video describes the technique of amplifying single-stranded DNA using oligonucleotide-coated polymer microspheres in a single-tube reaction. This process yields pure single-stranded DNA, which can be further used for DNA sequencing and microarray analysis.

This video describes nested polymerase chain reaction, a technique that consists of two sequential PCR amplification processes using two primer sets. The first set of primers is intended to anneal to sequences upstream of the second set, resulting in selective amplification of specific gene sequences. This PCR is more sensitive and specific than a normal PCR and is widely used as a detection technique for various diseases.

In this video, we describe a high-resolution melting PCR technique to determine the length polymorphism of DNA fragments of varying sequence lengths, based on melting temperature analysis.

This video demonstrates the multiplex droplet polymerase chain reaction technique for the simultaneous quantification of methylated DNA regions in different white blood cell populations as an early biomarker for clinical diagnosis and prognosis.

This video demonstrates the utility of digital PCR in a chip-in-a-tube format for the detection of single nucleotide variations in DNA samples. A single PCR reaction is partitioned into chambers that act as independent PCR reactions, and the detection of fluorescence signals from the amplified targets in the chambers is used to compute the frequency of the variant allele in the sample.

This video demonstrates chip-based digital PCR — a variation of the digital PCR technique that is useful in detecting rare transcript variants. The PCR reaction is partitioned into the chambers of a nanofluidic chip, each of which acts as an independent reaction. The detection of fluorescence signals from the chambers with amplified targets confirms the presence of rare transcript variants in the sample.