Final Exam Review

Recombinant DNA Technologies

Recombinant DNA

  • Definition: Recombinant DNA is DNA engineered from two different organisms. It is created by isolating a gene, joining it to DNA from another source, and cloning it to produce large quantities.
       - Quote: "Recombinant DNA (engineered from 2 different organism) technology… allows scientists to isolate a gene… join genes from different sources to create recombinant DNA…"

Homologous Recombination

  • Definition: Although not explicitly defined in the slides, it relates to homology-directed repair (HDR) in CRISPR, where template DNA with homology is used to repair a Cas9 cut.
       - Quote: "Alternatively a researcher can add template DNA for the correct gene… Homology directed repair (HDR)"

Restriction Enzymes & DNA Ligase

Restriction Enzymes

  • Definition: Enzymes that cleave DNA at specific sites.
       - Characteristics:
         - Recognize palindromic sequences.
         - Produce either:
           - 5’ sticky ends
           - 3’ sticky ends
           - Blunt ends.
       - Quote: "Restriction enzymes: Enzymes that cleave DNA at specific sites… recognize palindromic sequences… 5’ sticky ends, 3’ sticky ends, blunt ends."

DNA Ligase

  • Definition: Enzyme that joins DNA fragments by forming phosphodiester bonds.
       - Quote: "DNA ligase must be used to join the two annealed pieces of DNA together through the creation of a phosphodiester bond."

Plasmids as Vectors

  • Definition: Plasmids are circular DNA molecules used as vectors because they contain:
      - Origin of replication (ori): Allows self-replication.
      - Antibiotic resistance genes (e.g., ampicillin): Useful for selection.
      - lacZ gene: Used for blue/white screening.
      - Restriction enzyme cut sites: For insertion of genes.
      - Quote: "Plasmids are short circular pieces of DNA that contain: Origin of replication… Antibiotic resistance gene… Various restriction enzyme cut sites… lacZ."

  • Functionality:
      - Ampicillin resistance allows survival on ampicillin plates.
      - Disruption of lacZ produces white colonies when an insert is present, enabling selection.
      - Quote: "Selecting white colonies… allows us to select the bacteria that have our plasmid with the insert."

Palindromic Sequences

  • Definition: A palindromic sequence reads the same 5’→3’ on both strands of DNA.
       - Quote: "Nearly all restriction enzymes recognize palindromic sites. Sequences from 5’–3’ on each strand are the same."

Use of Restriction Enzymes in Recombinant Plasmids

  1. Cut the gene of interest with a restriction enzyme.

  2. Cut the plasmid with the same enzyme.

  3. Allow sticky ends to anneal.

  4. Use DNA ligase to seal the DNA backbone.
       - Quote: "A sticky end produced by EcoRI digestion can anneal… DNA ligase must be used to join the two annealed pieces…"

  • Bidirectional Cloning: Using two different enzymes prevents backward insertion.
       - Quote: "To remedy this you can cut the plasmid and the insert with two different restriction enzymes…"

Plasmid Insertion (Transformation & Electroporation)

Transformation

  • Definition: Introduction of plasmid DNA into bacteria.

  • Method: CaCl₂ treatment + heat shock improve the uptake.
       - Quote: "Treating bacteria with calcium chloride… heat shock… improves efficiency."

Electroporation

  • Definition: Application of an electrical pulse that opens membrane pores.

  • Applications: Works for both bacteria and animal cells.
       - Quote: "Electroporation… subjected to a brief electrical pulse that opens temporary holes… good way to introduce DNA into animal cells as well."

Applications of Recombinant DNA Technology

  • Uses:
      - Production of foods
      - Development of vaccines
      - Synthesis of antibiotics
      - Creation of vitamins
      - Production of proteins such as human growth hormone.
       - Quote: "Recombinant DNA technology can be used to make microbes produce… foods, vaccines, antibiotics, vitamins…"
       - Example: “Human growth hormone produced by genetically modified E. coli is a pure and cost-effective product.”

What is CRISPR?

  • Definition: A microbial adaptive immune system that employs RNA-guided nucleases to cut foreign DNA.
       - Quote: "CRISPR-Cas is a microbial adaptive immune system that uses RNA-guided nucleases to cleave foreign genetic elements."

  • Components:
      - Contains repeats, protospacer-derived sequences, and crRNA.
       - Quote: "These repeats are interspaced by short variable sequences… derived from exogenous DNA targets known as protospacers… make up the CRISPR RNA (crRNA) array."

CRISPR–Cas9 as a Genetic Engineering Tool

  • Function: Cas9 nuclease combined with sgRNA can target specific DNA near a PAM (Protospacer Adjacent Motif).
       - Applications:
         - Create mutations via Non-Homologous End Joining (NHEJ).
         - Insert correct DNA via Homology-Directed Repair (HDR).
         - Engineer eukaryotic cells.
         - Create transgenic animals.
       - Quote: "Cas9 has been used to generate engineered eukaryotic cells… Direct injection… enabled rapid generation of transgenic mice."

Functions of CRISPR Components

  • Cas9: Nuclease that induces double-strand breaks in DNA.
      - Quote: "Cas9 nuclease makes a double strand break…"

  • Guide RNA (sgRNA): A fusion of crRNA and tracrRNA that directs Cas9 to the target sequence.
      - Quote: "crRNA and tracrRNA are fused… to create a chimeric single-guide RNA (sgRNA)."

  • crRNA: Contains a 20-nucleotide guide sequence.
      - Quote: "Each crRNA unit contains a 20-nt guide sequence…"

  • tracrRNA: Required for processing of crRNA.
      - Quote: "tracrRNA… facilitates the processing of crRNA into discrete units."

  • PAM (Protospacer Adjacent Motif): A short sequence essential for Cas9 binding.
      - Quote: "Different CRISPR systems also require different protospacer adjacent motifs (PAMs)… allows CRISPR to cut."

NHEJ vs HDR

Non-Homologous End Joining (NHEJ)

  • Definition: Mechanism where cells reattach broken DNA ends.

  • Characteristics: Error-prone, leading to mutations.
       - Quote: "The cells DNA polymerase and ligase can reattach their ends. Non-homologous end joining (NHEJ)."

Homology-Directed Repair (HDR)

  • Definition: A researcher adds template DNA for precise correction of mutations.
       - Quote: "Alternatively a researcher can add template DNA… Homology directed repair (HDR)."

Current FDA-Approved CRISPR Medical Applications

  • Notes: The slides do not mention specific FDA-approved therapies.

  • General Reference: PEDIGREES AND INHERITABLE GENETIC DISEASES

Pedigrees & Genetic Disease Concepts

Pedigree

  • Definition: A diagram that tracks a genetic trait through multiple generations to help predict inheritance patterns.
       - Quote: "A pedigree traces a genetic trait through several generations and helps predict the future."

General Symbols Used in Pedigrees

  • Square = male

  • Circle = female

  • Shaded = affected

  • Unshaded = unaffected

  • Half-shaded = carrier (for recessive traits)

  • Horizontal line = mating

  • Vertical line = offspring

  • Diamond = sex unspecified

Autosomal Dominant vs Autosomal Recessive

Autosomal Dominant

  • Only one mutated allele needed to express the trait.

  • Appears in every generation.

  • Both sexes affected equally.

Autosomal Recessive

  • Requires two mutated alleles to express the trait.

  • Often skips generations.

  • Carriers are unaffected.

X-Linked Dominant vs X-Linked Recessive

X-Linked Dominant

  • One mutated allele on the X chromosome causes the disease.

  • Affected fathers pass the trait to all daughters and no sons.

X-Linked Recessive

  • Males with one mutated X chromosome are affected.

  • Females must have two mutated alleles to be affected.

  • More common in males.

RFLPs, VNTRs, STRs & Linkage Analysis

Restriction Fragment Length Polymorphisms (RFLPs)

  • Definition: Variations in DNA fragment sizes after digestion with restriction enzymes.
       - Quote: "The fragments in the digest are analyzed… The variations in fragment patterns are called restriction fragment length polymorphisms."

  • Detection of Mutations:
      - DNA is cut with restriction enzymes.
      - Mutations may create or destroy restriction sites, altering fragment sizes.
      - Gel electrophoresis reveals diverse patterns acting as markers linked to disease genes.
       - Note: RFLPs can determine haplotypes.

VNTRs and STRs

  • Definition: Highly polymorphic DNA markers used in linkage analysis.
       - VNTRs (Variable Number Tandem Repeats): Longer repeated sequences that vary in copy number.
       - STRs (Short Tandem Repeats): Shorter repeat units (2–6 bp) that vary between individuals.
       - Quote: "Depends on highly polymorphic DNA markers… RFLPs, VNTRs, STRs."

Common Inherited Diseases

Cystic Fibrosis

  • Gene Involved: CFTR gene.
       - Quote: "CF is… caused by two nonfunctional (or missing) copies of the CFTR gene."

  • Mutation Effects: Causes thick mucus, lung disease, pancreatic insufficiency, and high sweat chloride.
       - Note: CFTR protein regulates chloride transport.

  • Detection Methods:
      - 23-variant genotyping panel
      - NGS sequencing of CFTR
      - Copy number variant analysis.
       - Quote: "Can be diagnosed by genotyping 23… Can also be diagnosed by sequencing the CFTR gene using NGS."

Duchenne Muscular Dystrophy (DMD)

  • Gene Involved: DMD gene (largest human gene).
       - Quote: "The dystrophin gene is the largest known human gene… 79 exons."

  • Normal Gene Product: Dystrophin stabilizes muscle cell membranes.
       - Quote: "Dystrophin… strengthens muscle fibers and protects them from injury."

  • Mutation Effects: Most mutations are exon deletions (60–70%) causing frameshifts, leading to premature stop codons and lack of dystrophin.
       - Quote: "DMD is associated with mutations that disrupt the protein’s reading frame causing premature stop codons."

  • Detection Methods:
      1. Level 1: MLPA or microarray for deletions/duplications
      2. Level 2: NGS for small variants
      3. Level 3: RNA analysis (RT-PCR, RNA-seq).
       - Quote: "Three levels of testing are performed… MLPA… NGS… RNA-based methods."

Sickle Cell Anemia

  • Gene Involved: Hemoglobin beta chain gene (HBB).
       - Quote: "Sickle cell is caused by a mutation in the hemoglobin beta chain…"

  • Type of Mutation: A single base substitution (c.20A>T → p.Glu7Val).
       - Quote: "Glutamic acid is substituted with valine at position six."

  • Effects on Hemoglobin: Creates a hydrophobic patch on HbS which causes polymerization resulting in a sickle shape and obstructed blood flow.
       - Quote: "This hydrophobic site… allows for HbS to form long polymers creating the sickle shape."

  • Detection Methods:
      - PCR to detect the single base change.
       - Quote: "A simple PCR can detect the single base change…"

Huntington’s Disease

  • Gene Involved: HTT gene (IT15).
       - Quote: "The HTT gene… mutation… involves a CAG trinucleotide repeat."

  • Type of Mutation: CAG repeat expansion.
      - Normal: 10–35 repeats.
      - Disease: ≥40 repeats.
       - Quote: "In people with HD, it may repeat from 36 to over 120 times."

  • Effects on Gene Product: Produces abnormal huntingtin protein leading to neurodegeneration, movement disorders, and cognitive decline.
       - Quote: "Defective huntingtin protein leads to brain changes…"

  • Detection Methods:
      - PCR or Southern blot to measure repeat length.

Probe Based Techniques

Probe Definition

  • Definition: A probe is a single-stranded DNA or RNA sequence designed to hybridize with its complementary sequence in a genome.
       - Quote: "A probe is a single stranded sequence of DNA or RNA used to search for its complementary sequence…"

Possible Probe Labels

  • Fluorophores (fluorescent dyes)

  • Radioactive labels (e.g., ³²P)
       - Quote: "Probes contain a label… Fluorophore, P32."

Southern Blotting

Molecule Analyzed

  • Definition: DNA.
       - Quote: "A Southern blot is a method used to detect specific sequences in a sample of nucleic acid… Prepare DNA…"

General Steps of Southern Blotting

  1. DNA Preparation: Quantitate and normalize the DNA.
       - Quote: "First you must quantitate and normalize your DNA."

  2. Restriction Enzyme Digestion: Use a frequent cutter to fragment genomic DNA.
       - Quote: "Restriction enzymes… For Southern blot we must use a frequent cutter."

  3. Gel Electrophoresis: Run digested DNA on agarose gel producing a smear.
       - Quote: "After electrophoresis the gel will look like a smear…"

  4. Denaturation: Use NaOH or basic transfer buffer to convert dsDNA to ssDNA.
       - Quote: "Incubate gel in basic solution with NaOH to denature DNA."

  5. Transfer to Membrane (Capillary Transfer): DNA moves from gel to nylon membrane by capillary action.
       - Quote: "Wick transfer solution through gel onto nylon membrane via capillary action…"

  6. Fixing DNA: If basic transfer is not used, UV crosslinking is required.
       - Quote: "If basic conditions were used… we do not have to crosslink… If not we must fix… with UV light."

  7. Blocking: Treatment to prevent nonspecific probe binding using:
       - Salmon sperm DNA
       - SDS
       - Protein
       - Denhardt’s reagent.
       - Quote: "Membrane is then treated to block non-specific probe binding."

  8. Hybridization: Add denatured probe in hybridization buffer.
       - Note: Formamide lowers the melting temperature to keep DNA and probe denatured.
       - Quote: "Formamide… lower the melting temperature… keeping DNA and probe denatured."

  9. Stringency Washes: Remove unbound probe using appropriate conditions.
       - Quote: "High temp and low salt = increasing stringency…"

  10. Visualization: Use autoradiography for ³²P probes or fluorescence imaging for fluorescent probes.
        - Quote: "Autoradiography is used for 32P… Fluorescent light used for imaging fluorescently labelled probes."

Notes on Southern Blotting

  • Troubleshooting Issues:
       - Poor DNA quantitation leads to uneven band intensity and unreliable comparison.
       - Incomplete digestion results in large fragments and incorrect smear patterns.
       - Gel not denatured prevents probe binding resulting in no signal.
       - Poor transfer leads to weak or missing bands.
       - Inadequate blocking results in high background noise.
       - Incorrect stringency leads to loss of true signal or nonspecific binding.
       - Probe not denatured prevents hybridization.
       - Weak or smeared signals can arise from incorrect wash conditions.

  • Applications of Southern Blotting:
       - Detecting specific DNA sequences.
       - Detecting large repeat expansions (e.g., Fragile X syndrome).
       - Quote: "Important applications—disorders caused by large repeat expansions… ex. fragile X syndrome."

Northern Blotting

  • Purpose: Analyze RNA expression.
       - Quote: "In general it is a Northern blot done with RNA… No need to digest since RNA is already short."

FISH (Fluorescence In Situ Hybridization)

  • Purpose and Principle: Detect chromosomal abnormalities using fluorescent probes binding to specific DNA sequences within cells.
       - Quote: "FISH… identify chromosomal abnormalities… deletions, amplifications, translocations…"

  • Resolution: 1–5 kb.

Processing Blood Samples for FISH & Role of Colcemid

  • Process:
      - Grow cells and arrest in metaphase using colcemid.
      - Quote: "Colcemid added to arrest cells in metaphase."

  • Purpose of Colcemid: Stops mitosis for chromosome condensation and visualization.

Types of Probes Used in FISH

  1. Repetitive Sequence Probes: α-satellite, β-satellite, telomere repeats.

  2. Whole Chromosome Paint Probes.

  3. Unique Sequence Probes (1 kb–1 Mb).
       - Quote: "Types of probes: Repetitive sequence probes… Whole chromosome paint… Unique sequence probes."

Applications of FISH

  • Examples include:
      - BCR-ABL1 fusion (CML).
      - Solid tumors (breast, lung, sarcoma, glioma).
      - Rapid aneuploidy detection.
      - Microdeletion/microduplication syndromes (e.g., Williams syndrome).
      - Quote: "Applications—BCR-ABL1… solid tumors… aneuploidy… deletion/microduplication syndromes."

G-Banding & Karyotyping

G-Banding

  • Definition: A staining method to visualize chromosomes.
       - Quote: "G-banding… produce a visible karyotype… AT-rich heterochromatin stains dark… GC-rich euchromatin stains light."

Karyotyping

  • Process: Arranging chromosomes to detect:
      - Deletions
      - Duplications
      - Insertions
      - Translocations
      - Inversions
      - Aneuploidy.
       - Quote: "Chromosomes are visually examined to see if there is anything wrong…"
       

Aneuploidy

  • Definition: Abnormal number of chromosomes (extra or missing).

  • Detection: Can be identified using FISH and microarray.
       - Quote: "Applications… aneuploidy."

DNA Microarray

cDNA Microarray (Gene Expression)

  • Definition: Used to compare mRNA expression between samples.
       - Quote: "mRNA is isolated… reverse transcribed into cDNA… gene expression patterns."

Comparative Genomic Hybridization (aCGH)

  • Function: Detects copy number changes (deletions, duplications).
      - Patient DNA labeled with Cy3 and control DNA with Cy5.
      - Quote: "Sample –Cy3, Control –Cy5… Excess control = deletion… Excess patient = amplification."

Applications of Microarray

  • Used for:
      - Microdeletions / microduplications.
      - Aneuploidy.
      - Developmental delay.
      - Intellectual disability.
      - Autism.
      - Congenital abnormalities.
      - Oncology (brain tumors, neuroblastoma, hematologic cancers, sarcomas).

PCR

Principles of qPCR (Real-Time PCR)

  • Definition: qPCR (quantitative PCR or real-time PCR) is a modification of conventional PCR where the accumulation of PCR products is measured in real-time during each cycle.
       - Quote: "qPCR differs from conventional PCR in that the PCR product is detected in real-time as it is amplified."

How qPCR Works

  • Process: Fluorescent dyes or probes are added to the PCR master mix; as the denature-anneal-extend cycle progresses, fluorescence increases, which is detected by a special thermal cycler.
       - Quote: "We can observe the PCR in real time by incorporating fluorescent probes or dyes into the PCR master mix."

Types of Fluorescent Probes Used in qPCR

  • Categories:
      1. DNA Binding Dyes: Bind to any double-stranded DNA, fluorescence increases as more dsDNA is produced.
      2. Hybridization Probes: Fluorescent probes specifically binding to the target sequence.
      3. Hydrolysis Probes: TaqMan-type that binds to target and increases fluorescence when cleaved during extension.
      4. Hairpin Probes: Change in fluorescence when the hairpin structure opens upon binding to target DNA.

Threshold Cycle (CT) in qPCR

  • Definition: The thermal cycle at which the fluorescent signal becomes detectable is referred to as the cycle threshold (CT) value.
       - Quote: "The thermal cycle at which the fluorescent signal becomes detectable… is called the cycle threshold (CT) value."

  • Meaning:
      - Cycle number where fluorescence rises above background.
      - Inversely proportional to starting template amount.
       - Quote: "This value is inversely proportional to how much template you started with."

  • Interpretation:
      - Low CT indicates high starting DNA concentration.
      - High CT indicates low starting DNA concentration.

  • Calculation Note: CT calculated when fluorescence exceeds 10× the standard deviation of background.

Summary of qPCR Principles

  • Conclusion:
      - qPCR measures DNA amplification in real-time using fluorescent dyes or probes.
      - Types of probes include DNA-binding dyes, hybridization probes, hydrolysis probes (TaqMan-type), and hairpin probes.
      - Quote: "CT (cycle threshold)"