CSMLS Molecular Diagnostics 2 — Questions and Answers
Question 1: What is the function of restriction endonucleases in molecular biology?
- To amplify specific DNA sequences using thermal cycling
- To cleave double-stranded DNA at specific palindromic recognition sequences (Correct answer)
- To join DNA fragments with complementary ends
- To remove RNA primers from newly synthesized DNA
Correct 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.
Question 2: 16S rRNA gene sequencing is considered the gold standard for bacterial species identification because:
- It amplifies only pathogenic bacteria
- The gene contains conserved primer-binding regions flanking variable species-identifying regions (Correct answer)
- It detects antibiotic resistance genes directly
- It can identify bacteria without prior culture
Correct 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.
Question 3: In Southern blotting, why is the gel treated with alkali after electrophoresis?
- To stain DNA bands for direct visualization
- To denature double-stranded DNA into single strands before transfer (Correct answer)
- To neutralize the gel before probe hybridization
- To digest non-target DNA before transfer to the membrane
Correct 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.
Question 4: Which next-generation sequencing (NGS) platform uses fluorescently labeled reversible terminator nucleotides incorporated one base at a time?
- Pacific Biosciences SMRT sequencing
- Oxford Nanopore sequencing
- Illumina sequencing by synthesis (Correct answer)
- Ion Torrent semiconductor sequencing
Correct 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.
Question 5: Restriction fragment length polymorphism (RFLP) analysis detects genetic variation by:
- Measuring differences in gene expression between individuals
- Identifying sequence changes that create or abolish restriction enzyme recognition sites (Correct answer)
- Detecting chromosomal translocations with fluorescent probes
- Quantifying copy number variants by real-time PCR
Correct 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.
Question 6: Northern blotting is used to:
- Detect specific DNA sequences in genomic samples
- Detect and quantify specific mRNA transcripts in a sample (Correct answer)
- Identify specific proteins by antibody probing
- Determine the chromosomal location of a gene
Correct 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.
Question 7: Allele-specific PCR (AS-PCR) is designed primarily to:
- Amplify the entire coding sequence of a gene
- Detect specific single nucleotide variants or point mutations (Correct answer)
- Compare expression levels between two tissue types
- Identify gene fusions in hematological malignancies
Correct 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.
CSMLS Molecular Diagnostics 2
What is the function of restriction endonucleases in molecular biology?