Lecture 58 – Applications in Molecular Genetics I: Cytogenetics and Gene Amplification

What are the three main categories of genetic testing?

Detecting cytogenetic abnormalities, detecting known mutations, and detecting new mutations.

Define chromosomal karyotyping.

A cytogenetic technique used to analyze the number and morphology of nuclear chromosomes in a cell.

How are metaphase chromosomes grouped in karyotyping?

By size (largest to smallest) and centromere position (metacentric, submetacentric, acrocentric).

Which arm of a chromosome is shorter: p or q?

The p arm is short; the q arm is long.

What is the resolution of a standard G-banded karyotype?

Approximately 450–550 bands; detects changes >5–10 Mb (~150 genes).

List applications of karyotyping.

Detects aneuploidies (e.g., trisomies, Turner syndrome), large structural changes, and chromosomal translocations (e.g., Philadelphia chromosome).

What is G-banding?

Chromosome staining using trypsin and Giemsa; dark bands (heterochromatin) are gene-poor, light bands (euchromatin) are gene-rich.

Why are dividing cells required for G-banding?

Chromosomes must be in metaphase for visualization.

What does high-resolution chromosomal banding allow?

More detailed banding (550–850 bands) by arresting cells in prometaphase or prophase; detects smaller deletions.

When is high-resolution karyotyping indicated?

Unexplained congenital anomalies, developmental delay, and dysmorphic features.

Define fluorescence in situ hybridization (FISH).

A fluorescence-based technique for detecting and localizing specific DNA sequences on chromosomes using labeled probes.

What are the main steps of FISH?

Prepare and label probe → denature probe and target DNA → hybridize → visualize under fluorescent microscope.

What is the typical result pattern in FISH?

Each homologous chromosome pair shows two fluorescent dots for the target sequence.

What are the advantages of FISH over G-banding?

Higher sensitivity and resolution (~2 Mb); detects microdeletions, microduplications, and derivative chromosomes.

When is interphase FISH used?

For rapid (<24 h) diagnosis from nondividing cells such as amniotic fluid, tissue sections, or mucosal swabs.

List examples of aneuploidies detectable by interphase FISH.

Trisomy 21, 18, 13, and sex chromosome abnormalities (X/Y).

What is spectral karyotyping (SKY)?

A multicolor fluorescence method where each chromosome pair is uniquely labeled for automated karyotype analysis.

What are SKY’s applications?

Identifying marker chromosomes, complex rearrangements, and translocations.

Define array-based comparative genomic hybridization (aCGH).

A genome-wide assay comparing patient and reference DNA hybridized to microarray spots to detect copy number variations.

What does aCGH measure?

Relative copy number changes, but not chromosomal location or balanced rearrangements.

What resolution can aCGH achieve?

Down to 25–250 Kb.

Why should aCGH findings be confirmed by FISH or karyotyping?

Because aCGH cannot detect balanced rearrangements (e.g., inversions, translocations).

What is molecular cloning?

A recombinant DNA technique to amplify or express a specific DNA sequence in a host organism.

What enzyme is essential for molecular cloning?

Restriction endonucleases that cleave double-stranded DNA at specific palindromic sequences.

What are “sticky ends”?

Single-stranded overhangs generated by restriction enzymes to facilitate ligation of DNA fragments.

What is a cloning vector?

A DNA molecule capable of autonomous replication in a host cell (e.g., plasmid).

List key features of plasmid cloning vectors.

Origin of replication, restriction sites, and selectable markers (e.g., antibiotic resistance).

Give an example of a disease detected using restriction analysis.

Muenke syndrome, caused by a mutation in FGFR3 that creates a new restriction site.

Differentiate genomic vs. cDNA library.

Genomic library: includes entire genome (coding + noncoding). cDNA library: made from mRNA, includes only expressed genes.

What are recombinant expression systems used for?

Producing large amounts of human proteins (e.g., insulin, growth factors) using bacterial, yeast, or mammalian cells.

Define polymerase chain reaction (PCR).

A technique to exponentially amplify DNA from a small clinical sample using thermostable DNA polymerase.

List the main steps of PCR.

Denaturation (strand separation) → annealing (primer binding) → extension (DNA synthesis).

Which enzyme is critical for PCR?

Taq polymerase or another thermostable DNA polymerase.

How many PCR cycles are typically run?

~30–35 cycles, yielding billions of copies of the target sequence.

What is required to perform PCR?

Template DNA, primers, dNTPs, buffer, and thermostable polymerase.

How are PCR products analyzed?

By agarose gel electrophoresis, separating DNA fragments by size.

Which electrode does DNA migrate toward in gel electrophoresis?

Positive electrode (anode), since DNA is negatively charged.

What is quantitative real-time PCR (qPCR)?

A PCR technique that monitors amplification in real time to measure DNA quantity.

Differentiate qPCR and RT-PCR.

qPCR quantifies DNA; RT-PCR uses reverse transcriptase to convert RNA into cDNA for amplification.

List clinical applications of PCR.

Detection of infectious agents (e.g., viral genomes in blood), genetic mutations, and forensic DNA profiling.

What are advantages of PCR-based methods?

Rapid, highly sensitive, requires minimal sample material.