Laboratory techniques

  • Polymerase chain reaction
    • Molecular biology lab procedure used to amplify a desired fragment of DNA. Useful as a diagnostic tool (eg, neonatal HIV, herpes encephalitis, blastomere biopsy).
    • Needed: source DNA template that includes target region to be amplified, as well as flanking sequence adjacent to the region. No need for entire sequence to be known.
    1. Denaturation—DNA is heated to ~95°C to separate the strands.
    2. Annealing—Sample is cooled to ~55°C. DNA primers, a heat-stable DNA polymerase (Taq), and deoxynucleotide triphosphates (dNTPs) are added. DNA primers anneal to the specific sequence to be amplified on each strand.
    3. Elongation—Temperature is increased to ~72°C. DNA polymerase attaches dNTPs to the strand to replicate the sequence after each primer.
    • Heating and cooling cycles continue until the DNA sample size is sufficient.
  • Reverse transcription polymerase chain reaction
    • RT-PCR) is used to detect and quantify levels of messenger RNA (mRNA) in a sample. Similar to regular PCR in that it uses sequence-specific primers, thermostable DNA polymerase, and a pool of deoxyribonucleoside triphosphates to amplify a DNA template.
    •  In RT-PCR, this template is generated by the action of reverse transcriptase on the mRNA sample, producing a complementary DNA (cDNA) strand that can then be amplified by PCR.  Because cDNA is complementary to the mRNA sequence, it contains the exons of a gene along with the 5′ and 3′ untranslated regions.
  • CRISPR/Cas9
    • A genome editing tool derived from bacteria. Consists of a guide RNA (gRNA) , which is complementary to a target DNA sequence, and an endonuclease (Cas9), which makes a single or double-strand break at the target site. Break imperfectly repaired by nonhomologous end joining (NHEJ) → accidental frameshift mutations (“knock-out”) , or a donor DNA sequence can be added to fill in the gap using homology-directed repair (HDR) .
    • Not used clinically. Potential applications include removing virulence factors from pathogens, replacing disease-causing alleles of genes with healthy variants, and specifically targeting tumor cells.
  • Blotting procedures
    • SNoW DRoP:
      • Southern = DNA
      • Northern = RNA
      • Western = Protein
  • Southern blot
    1. DNA sample is enzymatically cleaved into smaller pieces, which are separated on a gel by electrophoresis, and then transferred to a filter.
    2. Filter is exposed to radiolabeled single-stranded DNA probe that recognizes and anneals to its complementary strand.
    3. Resulting double-stranded, labeled piece of DNA is visualized when filter is exposed to film.
  • Northern blot
    • Similar to Southern blot, except that an RNA sample is electrophoresed. Useful for studying mRNA levels, which are reflective of gene expression.
  • Western blot
    • Sample protein is separated via gel electrophoresis and transferred to a membrane.
    • Labeled antibody is used to bind to relevant protein.
  • Southwestern blot
    • Identifies DNA-binding proteins (eg, c-Jun,c-Fos [leucine zipper motif]) using labeleddouble-stranded DNA probes.
  • Flow cytometry
    • Laboratory technique to assess size, granularity, and protein expression (immunophenotype) of individual cells in a sample.
    • Cells are tagged with antibodies specific to surface or intracellular proteins. Antibodies are then tagged with a unique fluorescent dye. Sample is analyzed one cell at a time by focusing a laser on the cell and measuring light scatter and intensity of fluorescence.
    • Data are plotted either as histogram (one measure) or scatter plot (any two measures, as shown). In illustration:
      • Cells in left lower quadrant ⊝ for both CD8 and CD3.
      • Cells in right lower quadrant ⊕ for CD8 and ⊝ for CD3. Right lower quadrant is empty because all CD8-expressing cells also express CD3.
      • Cells in left upper quadrant ⊕ for CD3 and ⊝ for CD8.
      • Cells in right upper quadrant ⊕ for CD8 and CD3 (red + blue→ purple).
    • Commonly used in workup of hematologic abnormalities (eg, paroxysmal nocturnal hemoglobinuria, fetal RBCs in mother’s blood) and immunodeficiencies (eg, CD4 cell count in HIV).
  •  Microarrays
    • 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.
    • 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.
  • Enzyme-linked immunosorbent assay
    • Immunologic test used to detect the presence of either a specific antigen (eg, HBsAg) or antibody (eg, anti-HBs) in a patient’s blood sample. Detection involves the use of an antibody linked to an enzyme. Added substrate reacts with enzyme, producing a detectable signal. Can have high sensitivity and specificity, but is less specific than Western blot.
    • Direct ELISA tests for the antigen directly, while indirect ELISA tests for the antibody (thus indirectly testing for the antigen).
    • Indirect ELISA is similar to Western Blot, however, in Western Blot the proteins are first separated by electrophoresis
  • Karyotyping
    • A process in which metaphase chromosomes are stained, ordered, and numbered according to morphology, size, arm-length ratio, and banding pattern (arrows in A point to extensive abnormalities in a cancer cell).
    • Can be performed on a sample of blood, bone marrow, amniotic fluid, or placental tissue.
    • Used to diagnose chromosomal imbalances (eg, autosomal trisomies, sex chromosome disorders).
  • Fluorescence in situ hybridization
    • Fluorescent DNA or RNA probe binds to specific gene site of interest on chromosomes (arrows in A point to abnormalities in a cancer cell, whose karyotype is seen above; each fluorescent color represents a chromosome-specific probe).
    • Used for specific localization of genes and direct visualization of chromosomal anomalies at the molecular level.
      • Microdeletion—no fluorescence on a chromosome compared to fluorescence at the same locus on the second copy of that chromosome
      • Translocation—fluorescence signal that corresponds to one chromosome is found in a different chromosome (two white arrows in A show fragments of chromosome 17 that have translocated to chromosome 19)
      • Duplication—a second copy of a chromosome, resulting in a trisomy or tetrasomy (two blue arrows show duplicated chromosomes 8, resulting in a tetrasomy)
  • Restriction fragment length polymorphism analysis
    • Restriction sites are cleaved by specific endonucleases and can be used to identify polymorphisms within variant alleles on gel electrophoresis
  • Molecular cloning
    • Production of a recombinant DNA molecule in a bacterial host.
    • Steps:
      1. Isolate eukaryotic mRNA (post-RNA processing) of interest.
      2. Add reverse transcriptase (an RNA-dependent DNA polymerase) to produce complementary DNA (cDNA, lacks introns).
      3. Insert cDNA fragments into bacterial plasmids containing antibiotic resistance genes.
      4. Transform (insert) recombinant plasmid into bacteria.
      5. Surviving bacteria on antibiotic medium produce cloned DNA (copies of cDNA).
  • Gene expression modifications
    • 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.
        • Random insertion—constitutive.
        • Targeted insertion—conditional.
  • Cre-lox system
    • Can inducibly manipulate genes at specific developmental points (eg, to study a gene whose deletion causes embryonic death).
    • RNA interference 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

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