Mutation Detection Techniques

Question 1

What is allele-specific PCR used to detect?

Answer 1

Specific single-nucleotide mutations or polymorphisms.

Question 2

What is methylation-specific PCR used for?

Answer 2

To detect DNA methylation patterns that may cause gene inactivation.

Question 3

What is the main advantage of MLPA (Multiplex Ligation-dependent Probe Amplification)?

Answer 3

It can detect multiple mutations or deletions in a single reaction.

Question 4

What does FISH (Fluorescence in situ Hybridisation) analyse?

Answer 4

Chromosome-level abnormalities, such as structural aberrations and numerical changes.

Question 5

What is the purpose of comparative genomic hybridization (CGH)?

Answer 5

To compare DNA from two samples and detect chromosomal gains or losses.

Question 6

How does Southern blotting work?

Answer 6

It separates DNA fragments by size using restriction enzymes, and detects specific sequences with labeled probes.

Question 7

What does droplet digital PCR offer over traditional PCR?

Answer 7

It allows for highly accurate quantification of DNA, even from very small amounts, by isolating individual DNA molecules into droplets.

Question 8

What is the main advantage of DNA microarrays?

Answer 8

They can simultaneously analyze thousands of genes for mutations or expression levels.

Question 9

What is the function of karyotyping?

Answer 9

To visualize the number and structure of chromosomes in a cell, identifying abnormalities.

Question 10

What kind of information can mass spectroscopy provide about DNA?

Answer 10

It can measure the mass of DNA fragments, predicting their base composition for mutation detection.

Question 11

What enzymes are used in methylation-specific PCR, and what is the key difference between them?

Answer 11

MspI and HpaII; MspI is methylation-insensitive, while HpaII is methylation-sensitive and cannot cut methylated DNA.

Question 12

In ARMS-PCR, what is the role of tetra primers?

Answer 12

They are designed to selectively amplify mutant or wild-type alleles by exploiting sequence differences at the primer binding site.

Question 13

What is the primary diagnostic use of MLPA in genetic testing?

Answer 13

Detecting copy number variations, such as gene deletions or duplications, in disorders with known genetic mutations.

Question 14

In Southern blotting, why is DNA denatured before transferring to a nitrocellulose membrane?

Answer 14

To convert the DNA into single strands, allowing it to hybridize with complementary labeled probes during detection.

Question 15

What is the significance of using capillary electrophoresis in MLPA?

Answer 15

It separates PCR products based on size, allowing precise identification of multiple amplified fragments in a single reaction.

Question 16

What is the key limitation of using FISH for genome-wide mutation scanning?

Answer 16

FISH is limited to detecting known, targeted regions or abnormalities, making it unsuitable for detecting unknown mutations across the genome.

Question 17

How does droplet digital PCR (ddPCR) achieve higher sensitivity compared to traditional quantitative PCR (qPCR)?

Answer 17

By partitioning the DNA into millions of droplets, ddPCR allows for individual reactions in each droplet, increasing accuracy in quantifying rare mutations.

Question 18

What type of mutations can comparative genomic hybridization (CGH) fail to detect, and why?

Answer 18

CGH cannot detect balanced chromosomal rearrangements, such as translocations or inversions, because there is no net gain or loss of genetic material.

Question 19

How does the Illumina GoldenGate assay differentiate between allele-specific and locus-specific oligonucleotides?

Answer 19

The allele-specific oligos hybridize at the SNP site, while the locus-specific oligo binds to a region downstream, allowing ligation and PCR amplification only when both are perfectly matched.

Question 20

What aspect of next-generation sequencing (NGS) presents a major challenge in clinical applications?

Answer 20

The vast amount of data generated, making the interpretation of novel variants difficult due to limited knowledge of their clinical significance.

Question 21

What are the primary advantages of using cell-free fetal DNA (cffDNA) in non-invasive prenatal testing (NIPT) compared to traditional methods like amniocentesis?

Answer 21

It reduces the risk to the fetus as it only requires a maternal blood sample, unlike amniocentesis, which is invasive.

Question 22

Why can archived prenatal samples, such as those from chorionic villus sampling (CVS), present challenges for genetic testing?

Answer 22

The fixation methods used for storage may affect the quality and integrity of DNA, complicating analysis.

Question 23

What is the main clinical risk associated with using RNA isolated from prenatal samples for mutation analysis?

Answer 23

Misdiagnosis in heterozygotes for nonsense mutations due to nonsense-mediated mRNA decay (NMD), which can degrade the mutated transcripts.

Question 24

How does preimplantation genetic diagnosis (PGD) differ from other prenatal testing methods like CVS or amniocentesis?

Answer 24

PGD is performed on embryos created via in vitro fertilization (IVF) before implantation, testing a single cell from an 8-cell embryo to ensure it is healthy before continuing development.

Question 25

What type of genetic abnormalities can chromosome microarray analysis detect in prenatal testing that standard karyotyping might miss?

Answer 25

It can detect smaller deletions and duplications of genetic material, which may not be visible in standard karyotyping.