Biotechnology - MCQ Practice Questions

Reverse transcriptase synthesizes complementary DNA (cDNA) from an RNA template in the first step of RT-PCR, enabling amplification of RNA targets like mRNA.

dPCR partitions the PCR reaction into numerous isolated compartments, allowing absolute quantification without requiring a standard curve, and provides higher precision than qPCR.

AFLP combines restriction enzyme digestion with PCR amplification of specific DNA fragments, generating polymorphic banding patterns useful for genetic mapping and strain differentiation.

STRs (Short Tandem Repeats) exhibit high variability between individuals due to differences in repeat numbers, making them ideal for forensic DNA profiling and paternity testing.

GC-rich sequences tend to form stable secondary structures (hairpins, loops), which can inhibit primer annealing and polymerase extension, reducing PCR efficiency.

AS-PCR uses primers with mismatches at the 3' end that only allow amplification when perfectly matched to the target allele, providing rapid and cost-effective mutation detection.

Nested PCR uses outer primers for the first amplification round and inner primers for the second round, significantly improving specificity and sensitivity for detecting low-copy pathogens.

Phi29 polymerase, from bacteriophage Phi29, has high processivity and 3' to 5' exonuclease activity, making it ideal for isothermal multiple displacement amplification of entire genomes.

High self-complementarity and primer-dimer formation should be avoided as they reduce PCR efficiency and specificity. Primers should have minimal secondary structure.

SYBR Green intercalates into double-stranded DNA and exhibits enhanced fluorescence, making it ideal for real-time monitoring of PCR amplification.

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

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

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.

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

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.

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

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

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

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.

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.