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AGMT practice questions

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Question 1Genetic Testing Technologies and Methodologies

Which enzyme is most commonly used to amplify DNA in a standard polymerase chain reaction?

  1. Restriction endonuclease
  2. DNA ligase
  3. Reverse transcriptase
  4. Taq DNA polymerase Correct answer.

Explanation

PCR relies on a thermostable DNA polymerase that survives repeated high-temperature denaturation cycles, and Taq polymerase from Thermus aquaticus is the classic example. Reverse transcriptase, ligase, and restriction enzymes serve other molecular functions.

Question 2Genetic Testing Technologies and Methodologies

In Sanger sequencing, what is the role of dideoxynucleotides (ddNTPs)?

  1. They label the template strand with a fluorescent tag
  2. They terminate chain elongation because they lack a 3'-hydroxyl group Correct answer.
  3. They serve as primers for the sequencing reaction
  4. They increase the rate of polymerase processivity

Explanation

ddNTPs lack the 3'-OH needed for the next phosphodiester bond, so incorporation halts the growing chain and produces fragments of defined lengths. The resulting ladder of terminated fragments reveals the sequence.

Question 3Genetic Testing Technologies and Methodologies

A quantitative real-time PCR assay uses a fluorescent probe that emits signal only when the target sequence is amplified. The cycle at which fluorescence crosses a defined threshold is called the:

  1. OD260
  2. Rf value
  3. Ct value Correct answer.
  4. Tm value

Explanation

The threshold cycle (Ct) is the PCR cycle where reporter fluorescence exceeds background, and it is inversely related to starting template quantity. Tm describes melting temperature, OD260 measures nucleic acid concentration, and Rf is a chromatography term.

Question 4Genetic Testing Technologies and Methodologies

Which technique separates DNA fragments by size using an electric field through a gel matrix?

  1. Flow cytometry
  2. Mass spectrometry
  3. Gel electrophoresis Correct answer.
  4. Southern blotting

Explanation

Gel electrophoresis drives negatively charged DNA through a porous matrix, so smaller fragments migrate farther than larger ones. The other techniques serve different analytical purposes.

Question 5Genetic Testing Technologies and Methodologies

What is the primary purpose of a primer in a PCR reaction?

  1. To cleave the target amplicon
  2. To denature the double-stranded template
  3. To provide a free 3'-hydroxyl for polymerase extension Correct answer.
  4. To proofread mismatched bases

Explanation

Primers are short single-stranded oligonucleotides that anneal to the template and supply the 3'-OH that DNA polymerase extends. Denaturation is achieved by heat, not primers.

Question 6Genetic Testing Technologies and Methodologies

Which method is designed to detect a specific DNA sequence after it has been separated by size and transferred to a membrane?

  1. Southern blot Correct answer.
  2. ELISA
  3. Western blot
  4. Northern blot

Explanation

Southern blotting uses labeled probes to hybridize to DNA fragments immobilized on a membrane. Northern blots target RNA, Western blots target protein, and ELISA detects antigens or antibodies.

Question 7Genetic Testing Technologies and Methodologies

Next-generation sequencing platforms that perform massively parallel sequencing typically require which step before sequencing?

  1. Restriction fragment length polymorphism digestion only
  2. Two-dimensional gel electrophoresis
  3. Sanger chain termination
  4. Library preparation with adapter ligation Correct answer.

Explanation

NGS workflows construct libraries by fragmenting DNA and ligating platform-specific adapters that enable immobilization and amplification on the sequencing instrument. The other options describe older or unrelated techniques.

Question 8Genetic Testing Technologies and Methodologies

Which molecule serves as the template in a reverse transcription reaction?

  1. RNA Correct answer.
  2. Lipid
  3. Double-stranded DNA
  4. Protein

Explanation

Reverse transcriptase copies an RNA template into complementary DNA (cDNA). This is the basis of RT-PCR and RNA sequencing workflows.

Question 9Genetic Testing Technologies and Methodologies

A laboratory wants to determine the exact number of CAG repeats in a patient's HTT gene. Which approach is most suitable for sizing the repeat tract precisely?

  1. Western blot of huntingtin protein
  2. Enzyme-linked immunosorbent assay
  3. Fluorescence in situ hybridization on metaphase chromosomes
  4. PCR amplification followed by capillary electrophoresis fragment analysis Correct answer.

Explanation

Fragment analysis resolves amplicon lengths to single-base resolution, allowing the repeat number to be counted. FISH, Western blot, and ELISA do not provide exact repeat sizing.

Question 10Genetic Testing Technologies and Methodologies

Which PCR modification would best allow detection of a specific point mutation using two allele-specific primers that differ at their 3' ends?

  1. Inverse PCR
  2. Long-range PCR
  3. Allele-specific PCR Correct answer.
  4. Methylation-specific PCR

Explanation

Allele-specific PCR exploits the inability of polymerase to extend a primer whose 3' terminal base mismatches the template, so amplification occurs only for the matched allele. The other variants address different targets.

Question 11Genetic Testing Technologies and Methodologies

A sample shows a heterozygous deletion on array comparative genomic hybridization (aCGH). What does the array detect?

  1. A single-base substitution
  2. A DNA methylation change
  3. A loss of copy number in the affected region Correct answer.
  4. A balanced translocation breakpoint

Explanation

aCGH measures relative copy number by comparing probe hybridization intensities between patient and reference DNA, so deletions and duplications are detected. Balanced rearrangements and point mutations do not change copy number.

Question 12Genetic Testing Technologies and Methodologies

Which sequencing strategy is most appropriate for detecting low-frequency somatic variants in a tumor sample?

  1. Standard Sanger sequencing
  2. Southern blotting
  3. Karyotyping
  4. Deep coverage targeted next-generation sequencing Correct answer.

Explanation

Somatic variants may be present in a small fraction of reads, so high-depth NGS increases the chance of detecting them above background error. Sanger sequencing has limited sensitivity for low-level mosaicism.

Question 13Genetic Testing Technologies and Methodologies

In a multiplex ligation-dependent probe amplification (MLPA) assay, what does a reduced peak height for a specific probe indicate?

  1. Increased sample DNA concentration
  2. A point mutation within the probe binding site
  3. A deletion of the probed sequence Correct answer.
  4. A duplication of the probed sequence

Explanation

MLPA quantifies relative copy number by comparing normalized probe peak heights; a roughly 50% reduction indicates a heterozygous deletion. Duplications increase peak height, and point mutations generally do not alter peak ratios.

Question 14Genetic Testing Technologies and Methodologies

Which property of DNA allows it to migrate toward the positive electrode during gel electrophoresis?

  1. The presence of uracil residues
  2. The negatively charged phosphate backbone Correct answer.
  3. The hydrophobic base stacking
  4. The hydrogen bonding between strands

Explanation

Phosphate groups give DNA a net negative charge at neutral pH, so it moves toward the anode. Base stacking, hydrogen bonding, and uracil content do not determine electrophoretic charge.

Question 15Genetic Testing Technologies and Methodologies

What is the main advantage of digital PCR over quantitative real-time PCR?

  1. Larger amplicon size capacity
  2. Elimination of the need for primers
  3. Absolute quantification without a standard curve Correct answer.
  4. Detection of protein-protein interactions

Explanation

Digital PCR partitions the reaction into many droplets or wells and counts positive partitions by Poisson statistics, giving absolute copy numbers without reference standards. It still requires primers and does not measure proteins.

Question 16Genetic Testing Technologies and Methodologies

Which technique would be used to determine whether a specific mRNA transcript is expressed in a tissue sample?

  1. Karyotyping
  2. Southern blotting
  3. Reverse transcription PCR Correct answer.
  4. Restriction fragment length polymorphism

Explanation

RT-PCR converts mRNA to cDNA before amplification, so it detects expressed transcripts. Southern blotting and RFLP analyze DNA, and karyotyping examines chromosomes.

Question 17Genetic Testing Technologies and Methodologies

A clinician requests testing for a large, multi-exon deletion in the DMD gene. Which method is best suited to detect this type of copy-number change?

  1. Sanger sequencing of a single exon
  2. Serum protein electrophoresis
  3. Chromosome banding at 400-band resolution
  4. Multiplex ligation-dependent probe amplification Correct answer.

Explanation

MLPA quantifies exon-level copy number and is a standard approach for detecting multi-exon deletions or duplications. Sanger sequencing covers only the targeted region, and banding lacks the resolution for single-gene exon losses.

Question 18Genetic Testing Technologies and Methodologies

Which feature distinguishes pyrosequencing from Sanger sequencing?

  1. Use of dideoxynucleotide chain terminators
  2. Reliance on restriction enzyme digestion
  3. Detection of released pyrophosphate during nucleotide incorporation Correct answer.
  4. Separation of fragments by capillary electrophoresis

Explanation

Pyrosequencing detects light emitted when pyrophosphate released by nucleotide incorporation drives a luciferase reaction. Sanger sequencing instead uses chain-terminating ddNTPs and capillary separation.

Question 19Genetic Testing Technologies and Methodologies

Which of the following best describes the purpose of bisulfite conversion in DNA methylation analysis?

  1. It fragments DNA into uniform lengths
  2. It converts unmethylated cytosine to uracil while leaving methylated cytosine unchanged Correct answer.
  3. It adds fluorescent labels to methylated bases
  4. It removes methyl groups from all cytosines

Explanation

Bisulfite treatment deaminates unmethylated cytosine to uracil, which reads as thymine after amplification, whereas methylated cytosine is protected. This sequence difference allows methylation status to be inferred.

Question 20Genetic Testing Technologies and Methodologies

A laboratory performs karyotyping on a peripheral blood sample. Which cell stage is analyzed to obtain the clearest chromosome morphology?

  1. Metaphase Correct answer.
  2. Prophase
  3. S phase
  4. Telophase

Explanation

Chromosomes are most condensed and individually distinguishable at metaphase, making it the standard stage for banding analysis. Other stages show less condensed or dividing chromatin.

Question 21Genetic Testing Technologies and Methodologies

Which technique directly visualizes the location of a specific DNA sequence on a metaphase chromosome spread?

  1. Fluorescence in situ hybridization Correct answer.
  2. Sanger sequencing
  3. Quantitative PCR
  4. Western blotting

Explanation

FISH uses a fluorescently labeled probe that hybridizes to its complementary target on the chromosome, revealing its physical location. The other techniques do not provide spatial mapping on chromosomes.

Question 22Genetic Testing Technologies and Methodologies

A patient is being tested for a known familial point mutation. Which approach offers the fastest targeted result with the simplest interpretation for a single nucleotide change?

  1. Whole-genome sequencing
  2. Southern blotting
  3. Karyotype analysis
  4. Targeted genotyping assay such as a TaqMan allelic discrimination assay Correct answer.

Explanation

A targeted allelic discrimination assay uses allele-specific probes to genotype a known variant quickly and with straightforward interpretation. Whole-genome sequencing is broader and slower, and karyotyping or Southern blotting do not resolve point mutations.

Question 23Genetic Testing Technologies and Methodologies

In a PCR reaction, a primer dimer artifact is suspected. Which change is most likely to reduce its formation?

  1. Adding more template DNA without adjusting primers
  2. Decreasing the primer concentration to zero
  3. Increasing the number of PCR cycles
  4. Increasing the annealing temperature Correct answer.

Explanation

Primer dimers form when primers anneal to each other at low stringency, so raising the annealing temperature reduces nonspecific primer interactions. Increasing cycles or template can worsen or fail to resolve the problem.

Question 24Genetic Testing Technologies and Methodologies

A sequencing run reports a variant with a low mapping quality and strand bias. What is the most appropriate interpretation?

  1. The variant should be reported without review
  2. The variant is a benign polymorphism
  3. The variant is definitively pathogenic
  4. The variant call is suspect and requires orthogonal confirmation Correct answer.

Explanation

Low mapping quality and strand bias indicate possible alignment or artifact problems, so the call should be treated cautiously and verified by an independent method. Neither pathogenicity nor benign status can be concluded from these quality flags alone.

Question 25Genetic Testing Technologies and Methodologies

A laboratory needs to detect a known fusion transcript in an RNA sample. Which method is most direct?

  1. ELISA of the fusion protein
  2. Karyotyping alone
  3. Southern blotting of genomic DNA
  4. Reverse transcription PCR with primers spanning the fusion junction Correct answer.

Explanation

RT-PCR converts the fusion transcript to cDNA and primers flanking the junction amplify only the fused message. Southern blotting and karyotyping examine DNA or chromosomes, and ELISA measures protein rather than transcript.

Question 26Genetic Testing Technologies and Methodologies

Which enzyme is commonly used to amplify a target DNA sequence in a polymerase chain reaction (PCR)?

  1. Reverse transcriptase
  2. Restriction endonuclease
  3. Taq DNA polymerase Correct answer.
  4. DNA ligase

Explanation

PCR relies on a thermostable DNA polymerase, such as Taq, to synthesize new DNA strands during the extension step. The other enzymes have different roles in molecular biology.

Question 27Genetic Testing Technologies and Methodologies

In Sanger sequencing, what is the purpose of including dideoxynucleotides (ddNTPs) in the reaction?

  1. To terminate chain elongation at specific bases Correct answer.
  2. To increase the rate of DNA synthesis
  3. To prevent secondary structure formation
  4. To label the DNA with a fluorescent dye

Explanation

ddNTPs lack a 3'-hydroxyl group, so their incorporation stops further extension, generating fragments of varying lengths that end at each base. This is the basis of chain-termination sequencing.

Question 28Genetic Testing Technologies and Methodologies

Which type of genetic variation is most readily detected by standard karyotyping?

  1. Trinucleotide repeat expansion
  2. Aneuploidy Correct answer.
  3. Microdeletion
  4. Single nucleotide polymorphism

Explanation

Karyotyping visualizes whole chromosomes and can detect numerical abnormalities such as aneuploidy, as well as large structural changes. Smaller variants require higher-resolution methods.

Question 29Genetic Testing Technologies and Methodologies

Which technique separates DNA fragments based on size using an electric field in a gel matrix?

  1. Southern blotting
  2. Gel electrophoresis Correct answer.
  3. DNA sequencing
  4. Polymerase chain reaction

Explanation

Gel electrophoresis drives DNA fragments through a gel under an electric field; smaller fragments migrate faster, allowing size-based separation. The other techniques serve different purposes.

Question 30Genetic Testing Technologies and Methodologies

A laboratory needs to quantify the copy number of a specific gene with high precision and without a standard curve. Which method is most appropriate?

  1. Digital PCR Correct answer.
  2. Quantitative real-time PCR
  3. Sanger sequencing
  4. Multiplex ligation-dependent probe amplification

Explanation

Digital PCR partitions the sample into many reactions, allowing absolute quantification by counting positive partitions, and does not require a standard curve. It offers high precision for copy number measurement.

Question 31Genetic Testing Technologies and Methodologies

Which of the following is a key advantage of next-generation sequencing (NGS) over Sanger sequencing for genetic testing?

  1. It produces longer reads
  2. It can detect large structural variants
  3. It requires no amplification
  4. It enables massively parallel sequencing of many fragments Correct answer.

Explanation

NGS platforms sequence millions of DNA fragments simultaneously, providing high throughput and enabling detection of variants across many genes or the whole genome. Sanger sequencing is limited to one fragment at a time.

Question 32Genetic Testing Technologies and Methodologies

Which method is most suitable for detecting a specific RNA sequence in a tissue sample while preserving spatial information?

  1. Western blotting
  2. Fluorescence in situ hybridization Correct answer.
  3. Reverse transcription PCR
  4. Northern blotting

Explanation

FISH uses labeled probes to detect specific RNA (or DNA) sequences in cells or tissue sections, allowing visualization of the location of the target within the tissue architecture. The other methods either lack spatial resolution or detect protein.

Question 33Genetic Testing Technologies and Methodologies

Which of the following best describes the principle of allele-specific oligonucleotide (ASO) hybridization?

  1. It measures the amount of fluorescence emitted during PCR amplification
  2. It relies on differential melting temperatures of perfectly matched versus mismatched probes Correct answer.
  3. It uses two primers that differ at their 3' ends to discriminate alleles
  4. It employs ligation of two probes that hybridize adjacent to each other

Explanation

ASO hybridization uses short probes that are perfectly complementary to one allele; under stringent conditions, a mismatch destabilizes the probe-target duplex, reducing signal. This allows discrimination between alleles.

Question 34Genetic Testing Technologies and Methodologies

A patient with a suspected microdeletion syndrome has a normal karyotype. Which technique would be most appropriate to detect a submicroscopic deletion?

  1. Sanger sequencing of the candidate gene
  2. Fluorescence in situ hybridization (FISH) with a locus-specific probe Correct answer.
  3. G-banded karyotyping
  4. Southern blotting

Explanation

FISH with a probe specific to the deleted region can detect submicroscopic deletions that are below the resolution of karyotyping. It is commonly used for microdeletion syndromes.

Question 35Genetic Testing Technologies and Methodologies

Which of the following is a limitation of using short tandem repeat (STR) analysis for forensic identification?

  1. It cannot be used on degraded DNA samples
  2. It cannot be multiplexed
  3. It requires knowledge of the flanking sequences for primer design Correct answer.
  4. It is less discriminating than mitochondrial DNA analysis

Explanation

STR analysis relies on PCR amplification of repeat regions, which requires designing primers that bind to the conserved flanking sequences. If these sequences are unknown or mutated, amplification may fail.

Question 36Genetic Testing Technologies and Methodologies

In a quantitative real-time PCR (qPCR) assay, what does the cycle threshold (Ct) value represent?

  1. The cycle at which fluorescence exceeds a background threshold Correct answer.
  2. The melting temperature of the amplicon
  3. The number of cycles required to reach the exponential phase
  4. The maximum fluorescence intensity reached during the reaction

Explanation

The Ct value is the PCR cycle at which the fluorescence signal crosses a threshold, indicating the onset of exponential amplification. Lower Ct values correspond to higher initial target amounts.

Question 37Genetic Testing Technologies and Methodologies

A laboratory is validating a next-generation sequencing (NGS) panel for detecting germline variants. Which quality metric is most critical for ensuring that variants are detected with high sensitivity?

  1. Number of multiplexed samples
  2. GC content of the target regions
  3. Average read length
  4. Depth of coverage Correct answer.

Explanation

Depth of coverage (the number of reads covering a given base) directly affects the ability to detect variants; higher depth increases sensitivity, especially for heterozygous variants. Other factors influence quality but are not the primary determinant of sensitivity.

Question 38Genetic Testing Technologies and Methodologies

Which of the following best explains why bisulfite sequencing is used to assess DNA methylation?

  1. Bisulfite specifically methylates cytosines
  2. Bisulfite degrades methylated DNA
  3. Bisulfite induces mutations at methylated CpG sites
  4. Bisulfite converts unmethylated cytosines to uracil, which is read as thymine Correct answer.

Explanation

Bisulfite treatment deaminates unmethylated cytosine to uracil, which is converted to thymine during PCR, while methylated cytosines remain unchanged. Sequencing then reveals methylation status as a C/T difference.

Question 39Genetic Testing Technologies and Methodologies

A researcher is using CRISPR-Cas9 to create a knockout mouse model. Which component is essential for targeting the Cas9 nuclease to a specific genomic locus?

  1. A fluorescent reporter
  2. A guide RNA Correct answer.
  3. A repair template
  4. A restriction enzyme

Explanation

The guide RNA (gRNA) contains a sequence complementary to the target DNA, directing Cas9 to the correct location for cleavage. Other components may be used for specific applications but are not essential for targeting.

Question 40Genetic Testing Technologies and Methodologies

Which of the following is a major challenge in the clinical implementation of whole-genome sequencing for rare disease diagnosis?

  1. Limited coverage of coding regions
  2. Requirement for prior knowledge of the causative gene
  3. Inability to detect single nucleotide variants
  4. High cost and complexity of data analysis Correct answer.

Explanation

Whole-genome sequencing generates massive amounts of data, requiring sophisticated bioinformatics pipelines and significant resources for analysis and interpretation. It does not require prior knowledge of the gene and covers both coding and noncoding regions.

Question 41Molecular Genetics and Genomics

Which enzyme is primarily responsible for adding nucleotides to the growing DNA strand during replication in prokaryotes?

  1. Primase
  2. DNA ligase
  3. DNA polymerase III Correct answer.
  4. Helicase

Explanation

DNA polymerase III is the principal replicative polymerase in bacteria, extending the primer by adding deoxynucleotides to the 3' end. The other enzymes perform distinct roles such as strand separation, primer synthesis, or joining nicks.

Question 42Molecular Genetics and Genomics

In the genetic code, how many codons specify amino acids, excluding the stop codons?

  1. 64
  2. 20
  3. 61 Correct answer.
  4. 60

Explanation

Of the 64 possible triplet codons, three (UAA, UAG, UGA) are stop signals, leaving 61 sense codons that specify amino acids. The code is degenerate, so 61 codons map onto only 20 amino acids.

Question 43Molecular Genetics and Genomics

Which type of RNA carries amino acids to the ribosome during translation?

  1. rRNA
  2. snRNA
  3. mRNA
  4. tRNA Correct answer.

Explanation

Transfer RNA (tRNA) molecules are charged with specific amino acids and deliver them to the ribosome by pairing their anticodon with the mRNA codon. Ribosomal RNA forms the core of the ribosome, and mRNA carries the coding sequence.

Question 44Molecular Genetics and Genomics

Which bond connects the phosphate group of one nucleotide to the sugar of the adjacent nucleotide in a DNA strand?

  1. Hydrogen bond
  2. Phosphodiester bond Correct answer.
  3. Glycosidic bond
  4. Peptide bond

Explanation

Adjacent nucleotides in a DNA strand are joined by phosphodiester bonds linking the 3' hydroxyl of one sugar to the 5' phosphate of the next. Hydrogen bonds instead hold the two strands together between complementary bases.

Question 45Molecular Genetics and Genomics

Which process produces a mature mRNA in eukaryotes by removing introns and joining exons?

  1. Transcription
  2. Splicing Correct answer.
  3. Polyadenylation
  4. Capping

Explanation

Splicing removes intronic sequences and ligates exons to form the mature coding transcript. Capping and polyadenylation are separate processing events that modify the transcript ends.

Question 46Molecular Genetics and Genomics

Which of the following describes the phenotype of an individual carrying two identical alleles at a given locus?

  1. Hemizygous
  2. Compound heterozygous
  3. Homozygous Correct answer.
  4. Heterozygous

Explanation

Homozygosity means both alleles at a locus are the same. Heterozygosity means the two alleles differ, and hemizygosity means only one copy is present.

Question 47Molecular Genetics and Genomics

Which DNA repair mechanism corrects mismatched base pairs that escape proofreading during replication?

  1. Base excision repair
  2. Homologous recombination
  3. Nucleotide excision repair
  4. Mismatch repair Correct answer.

Explanation

Mismatch repair recognizes and removes incorrectly paired bases that remain after replication proofreading, using strand-discrimination signals to identify the newly synthesized strand. Other pathways address different lesion types.

Question 48Molecular Genetics and Genomics

What is the role of a promoter in gene expression?

  1. It encodes the amino-terminal signal peptide
  2. It terminates transcription by forming a hairpin
  3. It marks the transcript for degradation
  4. It binds RNA polymerase and defines the transcription start site Correct answer.

Explanation

A promoter is a DNA sequence upstream of a gene where RNA polymerase and associated factors assemble to initiate transcription at a defined start site. Termination and degradation signals reside elsewhere in the transcript.

Question 49Molecular Genetics and Genomics

A double-stranded DNA molecule contains 30% adenine. What percentage of its bases are guanine?

  1. 70%
  2. 30%
  3. 40%
  4. 20% Correct answer.

Explanation

By Chargaff's rules, adenine equals thymine, so A + T = 60%, leaving 40% for G + C. Since guanine equals cytosine, guanine is half of 40%, or 20%.

Question 50Molecular Genetics and Genomics

A couple who are both carriers of an autosomal recessive disorder have a child. What is the probability the child is affected?

  1. 50%
  2. 75%
  3. 25% Correct answer.
  4. 0%

Explanation

Two carrier parents (Aa x Aa) produce offspring in a 1:2:1 ratio of AA:Aa:aa, so one quarter are homozygous recessive and affected. The 50% figure applies to carrier status, not to being affected.