5. GENE MUTATION

Question 1

What types of gene mutations can affect large segments of DNA and potentially alter protein function significantly?

Answer 1

Deletions, insertions, inversions, and translocations.

Question 2

How can point mutations impact protein sequences differently than large-scale mutations?

Answer 2

Point mutations affect one or a few base pairs and may or may not alter the encoded amino acid, while large mutations can significantly change protein sequences.

Question 3

What method is used to directly detect mutated bases and contextualize them with surrounding sequences in point mutation analysis?

Answer 3

Sequencing methods

Question 4

Why might next-generation sequencing (NGS) be limited in detecting certain mutation types?

Answer 4

NGS may struggle with structural chromosomal abnormalities and copy-number variations, which may require additional validation through Sanger sequencing or other methods.

Question 5

How do conservative and nonconservative substitutions differ in their impact on protein function?

Answer 5

Conservative substitutions replace an amino acid with one of similar properties, often minimally affecting function, while nonconservative substitutions replace with a biochemically different amino acid, potentially altering function

Question 6

What is a frameshift mutation, and how does it affect protein synthesis?

Answer 6

A frameshift mutation results from the insertion or deletion of nucleotides not in multiples of three, disrupting the reading frame and often leading to premature stop codons.

Question 7

How can the position of a point mutation in the coding region influence its effect on protein structure?

Answer 7

Mutations at the 5’ (amino-terminal) end typically cause more drastic changes than those at the 3’ end, possibly leading to complete loss of function.

Question 8

Why might a detected point mutation not necessarily lead to an altered phenotype?

Answer 8

The mutation could be silent or conservative, which might not change the amino acid sequence or significantly impact protein function.

Question 9

What are the advantages of using immunoassays in biochemical testing for protein or metabolite detection?

Answer 9

Immunoassays are high-throughput, flexible, and can detect specific target molecules like hormones, antibodies, or biomarkers.

Question 10

How do enzyme-linked immunosorbent assays (ELISAs) improve upon radioimmunoassays (RIAs)?

Answer 10

ELISAs use enzyme-linked detection instead of radioactive markers, eliminating radiation hazards while allowing detection of immunoglobulins and other analytes.

Question 11

What evolutionary advantage might frameshift-induced stop codons provide in the genetic code?

Answer 11

They may protect cells by halting the synthesis of potentially harmful, long nonsense proteins.

Question 12

Analyze how the use of monoclonal antibodies (mAbs) improved the quality of immunohistochemistry (IHC) compared to polyclonal antibodies.

Answer 12

he specificity of mAbs for single epitopes reduces nonspecific staining, providing clearer, higher-quality imaging compared to polyclonal antibodies that target multiple epitopes with variable specificity.

Question 13

How did the invention of the hybridoma technique by Köhler and Milstein in 1975 revolutionize antibody production for IHC?

Answer 13

It allowed for the production of identical monoclonal antibodies, which enhanced specificity and reduced background noise, thus improving the accuracy and consistency of IHC results.

Question 14

Compare the direct and indirect methods of antibody staining in terms of signal strength and speed in IHC.

Answer 14

Direct staining is faster but has limited signal intensity due to the lack of amplification, while indirect staining takes longer but provides stronger signals through secondary antibody amplification, increasing sensitivity.

Question 15

How does the process of antigen retrieval address the challenges posed by formalin fixation in IHC?

Answer 15

Antigen retrieval techniques like enzyme digestion or heating reverse formalin-induced epitope masking, thus allowing antibodies to bind effectively and improving staining results.

Question 16

Evaluate the impact of background blocking techniques on the accuracy of IHC results.

Answer 16

Background blocking with agents like hydrogen peroxide or serum proteins minimizes nonspecific binding and prevents interference, resulting in more accurate visualization of specific antigen-antibody interactions.

Question 17

Differentiate between the separation mechanisms of normal-phase and reverse-phase HPLC columns.

Answer 17

Normal-phase columns separate based on hydrophilicity, with lipophilic molecules eluting faster; reverse-phase columns separate based on hydrophobicity, causing lipophilic molecules to elute slower.

Question 18

Examine why gas chromatography (GC) is particularly suited for detecting volatile compounds.

Answer 18

GC uses an inert gas mobile phase and vaporizes the sample, making it ideal for separating and analyzing volatile compounds with varying interactions in the stationary phase.

Question 19

Analyze the advantages and limitations of UHPLC compared to traditional HPLC.

Answer 19

UHPLC offers higher resolution and faster analysis due to smaller particle sizes and faster flow rates, but it is less suitable for complex or unfiltered samples, which could clog the system.

Question 20

Compare the roles of different detectors in HPLC, such as UV light absorption and mass spectrometry.

Answer 20

UV light absorption detectors are used for compounds that absorb UV, whereas mass spectrometry detectors provide detailed molecular information, allowing for more accurate identification of complex mixtures.

Question 21

Explain how dual staining in IHC enhances the interpretation of tissue morphology and target localization.

Answer 21

Dual staining allows visualization of multiple targets within the same tissue sample by using different antibodies and substrates, providing a comprehensive view of tissue morphology and target interactions.

Question 22

How would you apply the concept of mass-to-charge ratio in mass spectrometry to identify an unknown molecule?

Answer 22

By analyzing the spectrum obtained from the mass spectrometer, you can compare the mass-to-charge ratios of detected ions to known standards to identify the molecule.

Question 23

In what way can electrospray ionization (ESI) be utilized in a clinical setting for protein analysis?

Answer 23

ESI can be applied to analyze protein mixtures from patient samples, allowing for the identification and quantification of specific proteins linked to diseases.

Question 24

Describe a scenario where matrix-assisted laser desorption/ionization (MALDI) would be effectively used for high-molecular-weight biomolecules.

Answer 24

MALDI can be applied in the analysis of large peptides or proteins in a research setting to determine their molecular weight and structure

Question 25

How might you implement the primer extension technique to detect mutations in a patient’s DNA sample?

Answer 25

You would design primers specific to the target region, perform the extension with complementary dideoxynucleotides, and analyze the resulting mass to identify mutations.

Question 26

Explain how single-strand conformation polymorphism (SSCP) can be applied to detect genetic mutations

Answer 26

By denaturing PCR products and analyzing their migration patterns in a gel, you can determine conformational differences that indicate mutations in the DNA sequence.

Question 27

In what situations would you choose polymerase chain reaction (PCR) amplification to enhance mutation detection?

Answer 27

PCR should be applied when working with limited or low-concentration DNA samples to ensure sufficient material for reliable mutation analysis

Question 28

How can the results from mutation scanning be interpreted in a clinical context?

Answer 28

You would apply clinical data and family history to interpret the significance of detected mutations, considering their potential impact on patient phenotype.

Question 29

How would you apply allele-specific oligomer hybridization (ASO) to test for mutations in BRCA1 or BRCA2 genes?

Answer 29

You would design synthetic probes specific to the normal and mutant sequences, amplify the target DNA via PCR, and then hybridize the samples to the immobilized probes under specific conditions to detect binding, indicating the presence of mutations.

Question 30

In what way can you implement melt-curve analysis (MCA) to distinguish between normal and mutated DNA sequences?

Answer 30

You would perform real-time PCR to generate amplicons, then gradually increase the temperature while monitoring fluorescence to analyze the melting temperatures (T_m) of the sequences, identifying differences in peaks that indicate mutations

Question 31

Describe how you would utilize heteroduplex analysis to identify mutations in PCR products

Answer 31

After denaturing and renaturing PCR products from a test sample and reference, you would run the mixture on a gel to observe migration patterns, where differences in banding would indicate the presence of mutations

Question 32

How can you apply high-density oligonucleotide arrays to enhance mutation detection in a clinical setting?

Answer 32

By fragmenting test DNA and hybridizing it to a high-density array with specific probes, you can simultaneously screen for multiple potential mutations or SNPs by analyzing the fluorescent signals from bound probes

Question 33

Explain how you would interpret results from high-resolution melt-curve analysis (HR-MCA) to detect sequence differences

Answer 33

You would analyze the fluorescence signals from the FRET probes at varying temperatures, where a drop in fluorescence at a specific T_m would indicate a mismatch, suggesting the presence of a mutation compared to the reference sequence.

Question 34

How can the design of primers in sequence-specific PCR (SSP-PCR) be modified to detect point mutations

Answer 34

By ensuring the 3′ end of the primer perfectly matches the nucleotide to be analyzed, allowing for the presence or absence of product to indicate the mutation’s presence or absence

Question 35

In what way does multiplexed SSP-PCR enhance the detection of mutations

Answer 35

It increases the length of the normal or mutant primers to create differently sized products, allowing simultaneous detection of multiple mutations from a single sample

Question 36

Describe how allelic discrimination with fluorogenic probes differentiates between normal and mutant sequences.

Answer 36

By using two probes labeled with different fluorophores, the hybridized probe is digested by polymerase, releasing the reporter dye and generating a fluorescence signal specific to either the normal or mutant sequence

Question 37

What is the role of restriction fragment length polymorphism (RFLP) analysis in mutation detection?

Answer 37

RFLP detects mutations by amplifying regions around the mutation and using restriction enzymes to cut the PCR product, identifying changes in fragment size or structure caused by mutations.

Question 38

How does nonisotopic RNase cleavage assay (NIRCA) utilize RNA in mutation detection

Answer 38

NIRCA amplifies DNA to produce RNA transcripts, which are then denatured and renatured; mismatches due to mutations form heteroduplexes that are cleaved by RNase enzymes, allowing for analysis of fragment sizes

Question 39

What is the process of the Cleavase assay and its advantages in mutation analysis?

Answer 39

The Cleavase assay uses a proprietary enzyme to cleave hybridized probes from test sequences; its advantages include short hands-on time and optional PCR amplification for various clinical diagnostic applications

Question 40

Given the nucleotide sequence ATGCGTCACGAATTA, how would you express a mutation where a T replaces a C at position 7?

Answer 40

c.7C>T; the “c.” indicates coding sequence.

Question 41

How can you represent a deletion of nucleotides 6 and 7 in the sequence ATGCGTCACGAATTA?

Answer 41

c.6_7del or c.6_7delTC.

Question 42

If an insertion of TA occurs between nucleotides 5 and 6 in the sequence ATGCGTCACGAATTA, what would be the notation?

Answer 42

c.5_6insTA.

Question 43

Describe how to indicate a duplication of nucleotides 4 and 5 in the sequence ATGCGTCACGAATTA

Answer 43

c.4_5dupCG.

Question 44

If TC at positions 6 and 7 is deleted and GACA is inserted in the sequence ATGCGTCACGAATTA, how would you denote this mutation?

Answer 44

c.6_7delTCinsGACA or c.6_7>GACA.

Question 45

How would you write an inversion of GCGTCAC starting at position 3 to position 9 in the reference sequence ATCACTGGAA?

Answer 45

c.3_9inv7.

Question 46

In the context of recessive diseases, how would you express two mutations occurring in different alleles of a gene?

Answer 46

c.[2357C>T]; [2378CdelA].

Question 47

How is a mutation at nucleotide 2357 described when the other chromosome exhibits loss of heterozygosity?

Answer 47

c.[2357G>A];[0].

Question 48

What notation would you use for a G>T mutation 5 nucleotides into intron 2 that starts after the 91st nucleotide of exon 2?

Answer 48

c.91 + 5G>T.

Question 49

If a protein sequence of methionine–arginine–histidine–glutamic acid–leucine is altered by substituting arginine (R) with serine (S), how would you express the mutation?

Answer 49

p.R2S.

Question 50

How would you denote a frameshift mutation affecting histidine residue at codon 3?

Answer 50

p.H3fs or p.H3fs*.