Molecular Diagnostics Flashcards

What is the function of restriction endonucleases in molecular biology?

Answer: To cleave double-stranded DNA at specific palindromic recognition sequences

Restriction endonucleases recognize short specific palindromic sequences and cut both DNA strands at or near those sites, producing defined fragments used in cloning, RFLP analysis, and restriction mapping.

16S rRNA gene sequencing is considered the gold standard for bacterial species identification because:

Answer: The gene contains conserved primer-binding regions flanking variable species-identifying regions

The 16S rRNA gene has conserved regions that allow universal primers to bind across all bacteria, flanking variable regions whose sequences differ enough between species to provide definitive identification.

In Southern blotting, why is the gel treated with alkali after electrophoresis?

Answer: To denature double-stranded DNA into single strands before transfer

Alkali denaturation converts the double-stranded DNA in the gel into single strands; single-stranded DNA is then transferred to a membrane where it can hybridize with a complementary labeled probe.

Which next-generation sequencing (NGS) platform uses fluorescently labeled reversible terminator nucleotides incorporated one base at a time?

Answer: Illumina sequencing by synthesis

Illumina's sequencing-by-synthesis uses fluorescently labeled, reversible terminator dNTPs; after each base is incorporated and imaged, the terminator is cleaved to allow incorporation of the next nucleotide.

Restriction fragment length polymorphism (RFLP) analysis detects genetic variation by:

Answer: Identifying sequence changes that create or abolish restriction enzyme recognition sites

RFLP detects sequence polymorphisms where a nucleotide change creates or destroys a restriction site, producing fragments of different sizes that are separated and visualized by electrophoresis.

Northern blotting is used to:

Answer: Detect and quantify specific mRNA transcripts in a sample

Northern blotting separates RNA by electrophoresis, transfers it to a membrane, and hybridizes it with a labeled probe to detect and estimate the size and abundance of specific mRNA molecules.

Allele-specific PCR (AS-PCR) is designed primarily to:

Answer: Detect specific single nucleotide variants or point mutations

AS-PCR uses a primer whose 3' end matches only the target allele; amplification occurs exclusively when the primer perfectly hybridizes to that variant, enabling sensitive detection of specific SNPs or mutations.