Biochemistry - Laboratory Techniques

Question 1

Polymerase chain reaction

• Definition
• Steps
• Agarose gel electrophoresis

Answer 1

• Definition
  
  • Molecular biology laboratory procedure used to amplify a desired fragment of DNA.
  • Useful as a diagnostic tool (e.g., neonatal HIV, herpes encephalitis).
• Steps
  
  1. Denaturation—DNA is denatured by heating to generate 2 separate strands
  2. Annealing—during cooling, excess premade DNA primers anneal to a specific sequence on each strand to be amplified.
  3. Elongation—heat-stable DNA polymerase replicates the DNA sequence following each primer.
  • These steps are repeated multiple times for DNA sequence amplification.
• Agarose gel electrophoresis
  
  • Used for size separation of PCR products (smaller molecules travel further)
  • Compared against DNA ladder.

Question 2

Southern blot

Answer 2

• A DNA sample is enzymatically cleaved into smaller pieces, electrophoresed on a gel, and then transferred to a filter.
• The filter is then soaked in a denaturant and subsequently exposed to a radiolabeled DNA probe that recognizes and anneals to its complementary strand.
• The resulting double-stranded, labeled piece of DNA is visualized when the filter is exposed to film.
• SNo_W_ DRo_P_:
  
  • Southern = DNA
  • Northern = RNA
  • Western = Protein

Question 3

Northern blot

Answer 3

• Similar to Southern blot, except that an **RNA **sample is electrophoresed.
• Useful for studying mRNA levels, which are reflective of gene expression.
• SNo_W_ DRo_P_:
  
  • Southern = DNA
  • Northern = RNA
  • Western = Protein

Question 4

Western blot

Answer 4

• Sample protein is separated via gel electrophoresis and transferred to a filter.
• Labeled antibody is used to bind to relevant protein.
• Confirmatory test for HIV after (+) ELISA.
• SNo_W_ DRo_P_:
  
  • Southern = DNA
  • Northern = RNA
  • Western = Protein

Question 5

Southwestern blot

Answer 5

• Identifies DNA-binding proteins (e.g., transcription factors) using labeled oligonucleotide probes.
• SNo_W_ DRo_P_:
  
  • Southern = DNA
  • Northern = RNA
  • Western = Protein

Question 6

Microarrays

• Definition
• Applications

Answer 6

• Definition
  
  • Thousands of nucleic acid sequences are arranged in grids on glass or silicon.
  • DNA or RNA probes are hybridized to the chip, and a scanner detects the relative amounts of complementary binding.
• Applications
  
  • Used to profile gene expression levels of thousands of genes simultaneously to study certain diseases and treatments.
  • Able to detect single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) for a variety of applications including genotyping, clinical genetic testing, forensic analysis, cancer mutations, and genetic linkage analysis.

Question 7

Enzyme-linked immunosorbent assay

• Definition
• Indirect ELISA
• ƒƒDirect ELISA
• Applications

Answer 7

• Definition
  
  • Used to detect the presence of either a specific antigen (direct) or a specific antibody (indirect) in a patient’s blood sample.
  • Patient’s blood sample is probed with either indirect ELISA or direct ELISA
  • If the target substance is present in the sample, the test solution will have an intense color reaction, indicating a (+) test result.
• Indirect ELISA
  
  • Uses a test antigen to see if a specific antibody is present in the patient’s blood
  • A secondary antibody coupled to a color-generating enzyme is added to detect the first antibody.
• ƒƒDirect ELISA
  
  • Uses a test antibody to see if a specific antigen is present in the patient’s blood
  • A secondary antibody coupled to a color-generating enzyme is added to detect the antigen.
• Applications
  
  • Used in many laboratories to determine whether a particular antibody (e.g., anti-HIV) is present in a patient’s blood sample.
  • Both the sensitivity and specificity of ELISA approach 100%, but both false-positive and false-negative results occur.

Question 8

Fluorescence in situ hybridization

Answer 8

• Fluorescent DNA or RNA probe binds to specific gene site of interest on chromosomes.
• Used for specific localization of genes and direct visualization of anomalies (e.g., microdeletions) at molecular level (when deletion is too small to be visualized by karyotype).
• Fluorescence = gene is present
• No fluorescence = gene has been deleted.

Question 9

Cloning methods

• Definition
• Steps

Answer 9

• Definition
  
  • Cloning is the production of a recombinant DNA molecule that is self perpetuating.
• Steps
  
  1. Isolate eukaryotic mRNA (post-RNA processing steps) of interest.
  2. Expose mRNA to reverse transcriptase to produce cDNA (lacks introns).
  3. Insert cDNA fragments into bacterial plasmids containing antibiotic resistance genes.
  4. Transform recombinant plasmid into bacteria.
  5. Surviving bacteria on antibiotic medium produce cDNA.

Question 10

Gene expression modifications

• Transgenic strategies in mice involve:
• Cre-lox system
• RNA interference (RNAi)

Answer 10

• Transgenic strategies in mice involve:
  
  • Random insertion of gene into mouse genome
  • Targeted insertion or deletion of gene through homologous recombination with mouse gene
    
    • Knock-_out_ = removing a gene, taking it out.
    • Knock-_in_ = inserting a gene.
• Cre-lox system
  
  • Can inducibly manipulate genes at specific developmental points (e.g., to study a gene whose deletion causes embryonic death).
• RNA interference (RNAi)
  
  • dsRNA is synthesized that is complementary to the mRNA sequence of interest.
  • When transfected into human cells, dsRNA separates and promotes degradation of target mRNA, “knocking down” gene expression.

Question 11

Karyotyping

Answer 11

• A process in which metaphase chromosomes are stained, ordered, and numbered according to morphology, size, arm-length ratio, and banding pattern.
• Can be performed on a sample of blood, bone marrow, amniotic fluid, or placental tissue.
• Used to diagnose chromosomal imbalances (e.g., autosomal trisomies, sex chromosome disorders).