Molecular Diagnostics & Genetics Quiz

Molecular Diagnostics & Genetics Key Terminology

• Agarose: Gel-forming polysaccharide derived from seaweed, used in various biological applications.
• Molecular Diagnostics: Techniques using DNA, RNA, or mRNA to identify or characterize diseases caused by infections or genetic abnormalities.
• Allele: Different forms of a gene occurring at a specific locus on a chromosome.
• Cell Nucleus: Contains the genetic material of the cell, housing DNA.
• Codon: A sequence of three nucleotide bases in mRNA that specifies an amino acid during protein synthesis.
• Diploid: Cells containing two complete sets of chromosomes, one from each parent.
• Epigenetics: Study of changes in gene activity regulation and expression not linked to DNA sequence alterations.
• Exon: Portions of a gene coding for proteins, which are translated during protein synthesis.
• Gene Expression: The process through which specific genes are activated to produce proteins, tightly regulated within the cell.
• Genetic Code: The set of rules defining how sequences of nucleotides correspond to specific amino acids in proteins.
• Genome: The complete set of DNA, including all of its genes, within an organism.
• Genotype: The genetic makeup of an individual, represented by the alleles at specific loci.
• Haploid: Cells, such as gametes, containing a single set of chromosomes, crucial for sexual reproduction.
• Haplotype: A group of alleles inherited together from a single parent on a single chromosome.
• Heterozygous: Having two different alleles at a particular genetic locus.
• Homozygous: Possessing two identical alleles at a specific locus.
• Intron: Non-coding sequences found between exons within a gene.
• Ligation: The process of joining two DNA molecule ends, often used in cloning.
• Mutations: Permanent alterations in the DNA sequence, which can affect gene function and expression.
• Penetrance: The likelihood that a specific genotype will lead to the expression of a particular phenotype.
• Phenotype: The observable traits or characteristics of an organism, resulting from the interaction of its genotype and environment.
• Polymorphism: Variations in the nucleotide sequence among individuals, contributing to genetic diversity.
• Proteins: Large biomolecules made up of amino acids that perform a wide array of functions within organisms.
• Protein Expression: Process where different proteins are generated in cells based on their specific functions.
• Proteomics: Comprehensive study of all the proteins expressed by an organism, providing insight into cellular functions.
• RFLP (Restriction Fragment Length Polymorphism): Variances in the sizes of DNA fragments produced by restriction enzyme digestion, useful in genetic analysis.
• Sequence: The specific order of nucleotide bases along a DNA strand, crucial for genetic encoding.
• STR (Short Tandem Repeats): Repeated short DNA sequences (2-5 base pairs) found throughout the genome, useful in genetic profiling.
• Telomere: Repetitive DNA sequences located at the ends of chromosomes, protecting them during cell division.
• Transcription: The process in which genetic information from DNA is copied into mRNA, facilitated by the RNA polymerase enzyme.
• Transcriptome: The complete set of RNA molecules transcribed in a specific cell, reflecting gene expression at a given time.

Molecular Diagnostics Application

• Molecular diagnostics are essential for disease detection, prediction, paternity, forensic analysis, gene therapy, and drug design.
• Key applications include identifying the cause of disease (diagnosis), predicting disease progression, and analyzing DNA for paternity and forensic purposes.

DNA Replication (Semi-Conservative)

• DNA replication involves the separation of strands by helicase enzyme.
• DNA polymerase facilitates the pairing of bases on each strand with new complementary bases.
• This process results in the production of two new DNA strands, each an exact duplicate of the original DNA.

The Chemical Basis of Heredity

• DNA serves as the biological "blueprint" for life, providing the necessary information for cellular functions.
• It allows cells to live, grow, differentiate, and replicate, ensuring both consistency and variability in genetic traits.

Genetic Units of Human DNA

• Human DNA consists of nuclear DNA (diploid genome) and mitochondrial DNA.
• Nuclear DNA is packed in 23 pairs of chromosomes, including 22 homologous pairs (autosomes) and 2 sex chromosomes (XX or XY).
• The human genome contains approximately 6 billion base pairs and around 30,000 genes.

Karyotype Analysis

• Karyotyping assesses the normality of chromosome number and structure.
• Chromosomes are numbered by size and the position of the centromere, with regions labeled as short arm (p) and long arm (q).
• A pattern is created based on staining intensity or fluorescence to identify chromosomal abnormalities.

Mitochondrial DNA

• Mitochondrial DNA (non-nuclear) contains 16,569 base pairs and 37 genes.
• It exhibits a higher mutation rate and possesses 128 naturally occurring polymorphisms.
• Transmission occurs solely through maternal inheritance.

DNA/RNA Structure

• Composed of two types of nitrogen bases: purines and pyrimidines.
• Purines: Adenine (A), Guanine (G) present in both DNA and RNA.
• Pyrimidines: Cytosine (C) and Thymine (T) found in DNA, with Uracil (U) replacing Thymine in RNA.

Base Pairing and DNA Double Helix

• Hydrogen bonds form between pyrimidines and purines, adhering to Watson-Crick base pairing rules: A=T (or A=U in RNA), G≡C.
• The DNA double helix is oriented in opposite directions with a 5' end (beginning) and a 3' end (end).
• Nucleotide bases pair to create "base pairs," ensuring specific pairing sequences for genetic stability.

Human Gene Structure

• Genes consist of exons separated by introns, with sequences encoding information for protein synthesis.
• Variations in sequences lead to the production of different proteins.

The Central Dogma of Molecular Biology

• Describes the flow of genetic information: DNA → RNA → Protein.
• Involves three processes: replication, transcription, and translation.

Translation and Genetic Code

• Each set of three nucleotides (codon) on mRNA corresponds to a specific amino acid.
• Codons determine the sequence of amino acids in a polypeptide chain, forming proteins.

Transcription

• mRNA is formed as a complementary sequence to the DNA template.
• In RNA, uracil (U) replaces thymine (T) found in DNA.
• mRNA processing involves:
  • Splicing: removing introns.
  • Capping: modifying the 5' end of mRNA.
  • Polyadenylation: adding adenine nucleotides to the 3' end, creating a poly-A tail.
• Introns remain in the nucleus while exons exit.

Molecular Techniques for DNA Isolation

• Basic steps in isolating DNA from clinical specimens include:
  • Separating white blood cells from red blood cells.
  • Lysing white blood cells to release DNA.
  • Denaturing or digesting proteins.
  • Separating contaminants from DNA.
  • Precipitating DNA if needed and resuspending in buffer.

DNA Isolation Methods

• Techniques include:
  • Liquid phase organic extraction (phenol/chloroform).
  • Liquid phase non-organic extraction.
  • Solid phase procedures for purification.

Chromosomal Crossover (Genetic Recombination)

• Occurs during prophase I of meiosis when paired chromosomes exchange DNA segments.
• Matching regions break and reconnect, leading to gene exchange.

Sample Source and Storage

• DNA can be isolated from any nucleated tissue, with blood collected in anticoagulants (EDTA preferred).
• Storage conditions for DNA:
  • In TE buffer at 4°C for weeks or at -20°C to -70°C for long-term preservation.
• RNA should be stored in RNase-free ultrapure water at -70°C.

Meiosis and Genetic Recombination

• Meiosis occurs during gamete formation, generating four haploid gametes from a diploid progenitor cell.
• Meiotic recombination frequently involves homologous chromosomes exchanging genetic material.
• Chromosomal crossover happens during prophase I of meiosis, where physical regions of paired chromosomes break and reconnect to form new gene combinations.

Molecular Techniques for DNA Isolation

• Basic steps in isolating DNA from clinical specimens include separating white blood cells (WBCs) from red blood cells (RBCs), lysing WBCs, denaturing digestive proteins, separating contaminants from DNA, and resuspending DNA in a buffer.
• DNA isolation methods include liquid phase organic extraction (phenol/chloroform), liquid phase non-organic extraction, and solid phase procedures.

Nucleic Acid Analysis

• Nucleic acid quantity and quality measurement techniques include UV spectrophotometry, agarose gel electrophoresis, fluorometry, and colorimetric blotting.
• DNA and RNA absorb light at 260 nm; proteins absorb at 280 nm, crucial for determining nucleic acid purity.
• For DNA and RNA quantitation:
  • DNA concentration = A260 × dilution factor × 50 µg/ml
  • RNA concentration = A260 × dilution factor × 40 µg/ml
• A260/A230 ratio is used as a measure of nucleic acid purity, with a ratio of 1.8–2.0 indicating high purity.

Restriction Endonucleases

• Bacterial enzymes that cut specific DNA sequences, referred to as "molecular scalpels."
• They recognize specific short recognition sequences (usually palindromes), often 4 to 6 bases long.

DNA Microarrays

• DNA microarrays use hybridization technology for examining gene expression across thousands of genes simultaneously.
• They play significant roles in gene expression studies, disease diagnostics, and pharmacogenetics.
• Special equipment is required for microarray generation and analysis.

Gene Cloning (Recombinant DNA Technology)

• Involves isolating and amplifying a defined DNA sequence using vectors (e.g., plasmids) and appropriate host organisms like E. coli.

DNA Sequencing Techniques

• Several methods for determining the nucleotide order in DNA:
  • Maxam-Gilbert (chemical degradation)
  • Sanger method (dideoxy chain termination)
  • Pyrosequencing (sequence by synthesis)
  • Next-generation sequencing (real-time sequencing of single DNA molecules) using technologies from Pacific Biosciences and Oxford Nanopore.

DNA Microarrays (DNA Chip)

• Utilizes hybridization technology for examining gene expression.
• DNA sequences are arranged on a solid support, enabling analysis.
• Each microarray contains thousands of genes to monitor expression levels simultaneously.
• Applications include gene expression studies, disease diagnosis, and pharmacogenetics (drug discovery).
• Requires specialized instrumentation for the creation and analysis of microarrays.
• Palindromic sequences remain the same when read in either direction (e.g., 5' - GGTACC - 3' and 3' - CCATGG - 5').

Gene Cloning (Recombinant DNA Technology)

• Focuses on isolating and amplifying specific DNA sequences.
• Primer annealing occurs at temperatures between 50-60°C.
• Polymerase chain reaction (PCR) involves extending primers at 72°C to synthesize full-length genes.
• Each PCR cycle doubles the amount of DNA, exponentially increasing genetic material.

Fluorescent In-Situ Hybridization (FISH)

• Involves the hybridization of a fluorescent DNA probe to complementary DNA in preserved tissues or cells.

Real-Time PCR / Quantitative PCR (qPCR)

• A dual qualitative and quantitative technique for detecting fluorescent reporters after each amplification cycle.

Nucleic Acid Sequence Based Amplification (NASBA)

• Amplification technique targeting RNA and rRNA specifically.
• Used for the direct detection of RNA viruses, such as HCV and HIV.

Strand Displacement Amplification (SDA)

• Amplification method using heat denaturation, annealing, and extension.
• Generates an altered target with restriction endonuclease recognition sites.

Branched Chain DNA (bDNA)

• Utilizes a series of hybrid probes to amplify signals through chemiluminescence.
• Enables detection of specific RNA sequences.

Electrophoresis of Nucleic Acids

• Method separates nucleic acids based on size and charge through agarose or polyacrylamide gel.
• DNA migrates toward the positively charged anode due to its negative charge.
• Migration rate depends on the gel's matrix type, nucleic acid charge, and size.

Factors Affecting Migration Rate

• Type and porosity of the gel, net charge of the molecule, DNA conformation, electric field strength, and temperature.
• Larger DNA fragments migrate slower due to difficulty navigating small gel pores, while smaller fragments move faster.

Blotting Techniques

• Southern Blot: Used to detect specific DNA sequences, where DNA is denatured in gel and transferred to a membrane by capillary action.
• High salt solutions facilitate the transfer of DNA to membranes for subsequent analysis.

Branched Chain DNA (bDNA)

• Utilizes hybrid probes for signal amplification via chemiluminescence.
• Designed for detecting specific RNA sequences.

Electrophoresis of Nucleic Acids

• Separation of nucleic acids based on size and charge using agarose or polyacrylamide gels.
• Migration rate is inversely proportional to the logarithm of molecular size (number of base pairs).
• Negatively charged DNA migrates towards the positively charged anode.

Factors Affecting Migration Rate

• Matrix type and gel porosity impact movement through the gel.
• The net charge of the nucleic acid influences migration speed.
• DNA conformation plays a role in how easily it can pass through gel pores.
• Strength of the electric field and gel temperature affect nucleic acid movement.
• Base composition and the presence of intercalating dyes can alter migration.

Pulsed Field Gel Electrophoresis (PFGE)

• Used for analyzing large DNA fragments up to 100 kilobases.
• Separation achieved by employing a pulsed electric field.
• Commonly applied in the genotyping of prokaryotes.

Southern Blot Technique

• Detects specific DNA sequences by transferring DNA from a gel to a membrane via capillary action in a high-salt solution.
• DNA is denatured in the gel using an alkaline solution.
• Utilizes a labeled complementary probe for the detection of target DNA.

Western Blot Technique

• Proteins are separated using SDS-polyacrylamide gel electrophoresis (PAGE).
• Proteins are transferred to membranes via electro-transfer.
• Membrane is incubated with a primary antibody, followed by a secondary antibody for visualization.
• Detection can be done using radioactive isotopes, chemiluminescent dyes, or colorimetric techniques.

Northern Blot Technique

• Detects specific RNA sequences by transferring RNA to a membrane using a high-salt solution.
• Labeled complementary probes are employed for detection.

Dot and Slot Blot Techniques

• Provide quick analysis of DNA and RNA without determining the size of the target.
• Applicable for expression, mutation, and amplification analyses.

Reverse Dot Blots

• Utilize allele-specific oligonucleotide hybridization for genotyping human mutations.

Stringency

• Refers to the conditions under which hybridization occurs, influencing the specificity of the detection process.

Human Genome and Genetics

• The human genome is diploid, consisting of 23 chromosome pairs, totaling approximately 6 billion bases.
• Chromosome structures are linear, not circular.
• Autosomal dominant traits have a 50% chance of being passed to offspring from a heterozygous parent.

Molecular Techniques and Principles

• Electrophoresis migration rates depend on net charge, size of the molecule, and thickness of the gel.
• Polymerase Chain Reaction (PCR) cycles include denaturation, annealing, and extension.
• Reverse Transcription PCR (RT-PCR) specifically requires RNA isolation and utilizes reverse transcriptase.

Genetic Terms

• A person with normal and mutant banding patterns is termed heterozygous; individuals with identical patterns are homozygous.
• Coding sequences of genes are referred to as exons, while non-coding regions are known as introns and are spliced out before translation.

Inheritance Patterns

• X-linked recessive traits are more common in males due to having only one X chromosome, while females may carry the trait but not express it due to having two X chromosomes.
• Autosomal recessive traits require two copies of the abnormal gene for expression and can skip generations.

Experimental Techniques

• Western blotting is a method used to detect proteins, while Southern blotting identifies DNA and Northern blotting detects RNA.
• Restriction endonucleases recognize specific palindromic DNA sequences.

Absorbance and DNA/RNA Quantification

• Absorbance readings at 260 nm are essential for calculating nucleic acid concentrations.
• For a 1:100 dilution giving an A260 of 0.430, the DNA concentration can be calculated accurately based on dilution factors.
• The spectrophotometric analysis of a DNA sample can determine total DNA contained based on dilution and absorbance readings.

SNP and Contaminants

• Single Nucleotide Polymorphisms (SNPs) can be determined through sequence variations in genotypes.
• Contaminants in DNA preparations can be indicated by spectrophotometric slight absorbance differences, such as contamination from chloroform or phenol.

Pedigree Analysis

• Pedigrees can illustrate inheritance patterns, helping identify whether traits are autosomal dominant, autosomal recessive, X-linked dominant or recessive, by tracking trait expression across generations.