Biotechnology

In multiplex PCR, primers with similar Tm values ensure equal amplification efficiency across all targets. Amplicons of comparable sizes prevent preferential amplification of shorter fragments (which amplify faster), ensuring balanced and accurate STR profiles for individual identification.

The optimal annealing temperature should be set at or slightly below the Tm of the primer with the lowest Tm (58°C) to prevent primer dissociation while maintaining specificity. Using 58°C ensures both primers can bind efficiently.

NGS requires substantial DNA; PCR amplifies adaptor-ligated fragments while incorporating index sequences for multiplexing, enabling efficient library preparation.

PCR-DGGE separates PCR amplicons based on melting behavior, providing a fingerprint of microbial community structure independent of culture.

Heterozygotes carry both alleles; separate allele-specific primer sets each amplify their respective allele, showing amplification in both reactions.

STR profiling amplifies variable number tandem repeats (VNTRs) and is the gold standard for DNA fingerprinting in forensics, recognized by Indian law enforcement and courts.

Ct is the cycle at which fluorescence surpasses background, inversely correlating with initial template amount. Lower Ct = higher initial DNA quantity.

Conventional PCR provides only end-point detection via gel electrophoresis, lacking real-time kinetic data. qPCR enables quantification during amplification and reduces post-PCR work.

AFLP uses a rare cutter (EcoRI) and frequent cutter (MseI) to generate polymorphic fragments that are amplified using selective PCR.

Degraded DNA has broken fragments; amplifying short segments (<200 bp) increases success rate compared to long-range PCR which requires intact template.