ICAR AIEEA PG 2024 - Plant Biotechnology

In the standard Gubler and Hoffman method for double-stranded cDNA synthesis, the first strand is synthesized from mRNA by reverse transcriptase (such as AMV-RT or M-MuLV RT). RNase H specifically cleaves the phosphodiester bonds of the RNA strand in an RNA:DNA hybrid, generating small RNA fragments. These residual RNA fragments act as primers for E. coli DNA polymerase I to synthesize the second cDNA strand via nick-translation, after which DNA ligase seals any remaining nicks.

According to Beer-Lambert's law, nucleic acids absorb ultraviolet light maximally at $260 \text{ nm}$ due to the conjugated ring systems of purine and pyrimidine bases. By standard convention:
$1.0 \; A_{260} \text{ unit} = 50 \; \mu\text{g/mL}$ for double-stranded DNA (dsDNA)
$1.0 \; A_{260} \text{ unit} = 40 \; \mu\text{g/mL}$ for single-stranded RNA (ssRNA)
$1.0 \; A_{260} \text{ unit} = 33 \; \mu\text{g/mL}$ for single-stranded oligonucleotides/ssDNA.

In standard constant-field agarose gel electrophoresis, DNA molecules larger than $30 - 50 \text{ kb}$ migrate together regardless of size (reptation limit). Pulsed-Field Gel Electrophoresis (PFGE), developed by Schwartz and Cantor, periodically alternates the direction of the electrical field across the gel. Larger DNA molecules require more time to reorient each time the electric field changes direction, allowing the resolution of very large DNA molecules ranging from $50 \text{ kb}$ up to $10 \text{ Mb}$ (intact fungal chromosomes, YACs, and bacterial genomes).