Laboratory methods

Overview of blotting techniques

Blotting is a technique used to detect DNA, RNA, and proteins. There is a variety of blotting techniques, with western, northern, and southern blot being the ones most commonly used in medical practice. All blotting techniques are based on the basic procedures/principles:

  1. DNA, RNA, or protein molecules contained in the sample are seperated by gel electrophoresis depending on their size and/or electric charge.
    • Small molecules travel faster than bigger molecules on gel electrophoresis.
  2. The seperated molecules are transferred from the gel onto a membrane. This transfer is called “blotting”.
  3. The DNA, RNA or protein molecule of interest is detected by incubating the membrane with a labeled, highly specific oligonucleotide probe or antibody, respectively.
    • The specificity of the probe is guaranteed by designing it complementary to the nucleotide sequence to be detected and that it binds solely to this sequence.
    • Monoclonal antibodies are used for the detection of the protein of interest in Western blot.
  4. To visualize the probe or the antibody, different techniques are used, e.g. autoradiography or (more commonly) detection of fluorescent dyes covalently linked to the probe or the antibody.

Comparison of blotting techniques

Western blot Northern blot Southern blot
Sample
Detection
  • Direct or indirect detection using
  • 32P-DNA or -RNA
  • DNA or RNA labeled with chemiluminescent dye
  • 32P-DNA
  • DNA labeled with chemiluminescent dye
Uses (examples)


SNoW DRoP:
S-outhern blot = D-NA
N-orthern blot = R-NA
o o
W-estern blot = P-roteins

Western blot (immunoblot)

Northern blot

  • Sample: RNA
  • Procedure
    • The RNA sample is cleaved by enzymes and separated by gel electrophoresis (commonly on agarose gels).
    • Separated and cleaved RNA is transferred to a membrane.
    • The membrane is incubated with labeled probes of RNA or DNA.
      • RNA probes recognize and the complementary strand if it is present on the membrane.
  • Result: double-stranded RNA; one unlabeled strand (cleaved RNA sample); one labeled strand that can be visualized when the membrane is exposed to a film
  • Uses

References:[1]

Southern blot

Enzyme-linked immunosorbent assay (ELISA)

Overview

ELISA is an assay technique that is often used as a diagnostic test to detect and quantify proteins (e.g., tumor markers, viral proteins, drugs, antibodies). The detection method is based on the highly specific interaction between an antibody and its antigen (i.e., the protein of interest). The antigen is immobilized on a microtiter plate and is bound by an enzyme-coupled antibody. The enzyme catalyzes a reaction when incubated with its substrate that is chromogenic, chemifluorescent or chemiluminescent. The intensity of the signal is directly proportional to the amount of captured antigen.

Direct ELISA

  1. The patient's sample supposedly containing the protein of interest (i.e. the antigen) is added to a well of microtiter plates with a buffered solution.
  2. The specific antibody-enzyme conjugate is added to the solution.
  3. A substrate for the enzyme is added
  4. Spectrometry is used to detect the generated chromophore
    • Higher concentration of antibodies binding to the antigen: stronger signal
    • Lower concentration of antibodies binding to the antigen: weaker signal

Indirect ELISA

Sandwich ELISA

  1. A surface plate is coated with capture antibodies (not the patient's antibodies).
  2. The sample is added to the plate.
    • Antigens present will be captured by capture antibodies coating the surface plate.
  3. Specific (labeled) antibodies for the antigen are added.
    1. If the antigen is present, the antibody binds to the antigen.
    2. Capture antibody-antigen-added (labeled) antibody” makes up the sandwich
  4. A substrate for the enzyme is added (color, fluorescent, or electrochemical changes are due to the reaction between substrates and enzymes).
  5. Spectrometry, fluorescence, or electrochemical studies are performed to assess for the amount of antigens present.

Uses

References:[2]

Polymerase chain reaction (PCR)

Polymerase chain reaction (PCR) is a method of amplifying fragments of DNA. This technique allows for segments of specific chromosomes to be amplified more than a million-fold and testing of very small sequences of DNA that could not otherwise be studied. A PCR usually consists of 25–50 cycles, which are divided into phases: denaturation; of the double helix, primer hybridization (to refine the target region), and elongation and amplification of the target region. Common sequences of DNA studied by PCR include: mutations, microsatellite instability sequences, and short tandem repeats (STRs).

  1. Denaturation
    • Complementary primers are added to DNA sample (primer pair selects the region of DNA to be amplified).
      • Primers are added in excess (this allows for primers to anneal with DNA strands, rather than annealing of previously separated DNA strands with each other).
    • Addition of enzymes (for DNA synthesis); : heat-stable DNA polymerase (Taq DNA polymerase)
    • Deoxyribonucleotides (dNTPs)
    • Heating of the sample separates the two DNA strands of the double-stranded DNA and yields single-stranded DNA.
      • Target temperatures: 90–98°C (194–208°F) for 20–30 seconds
      • Breaks hydrogen bonds between complementary base pairs
  2. Hybridization (annealing)
    • Cooling of the sample
      • Target temperature: 50–65°C (122–149°F) for 20–40 seconds
      • Primers bind to 3' ends of the DNA sequence to be amplified
  3. Elongation and amplification
    • Heating of the sample
      • Target temperature: 70–80°C (158–176°F); 72°C (162°F) is most commonly used for DNA polymerase
    • Repetition of the process (approximately 25–50 cycles); → yields an amplification to 106 - 1010 copies.

References:[1]

Chromosome testing

Molecular biological methods are used in the (prenatal) diagnosis of heritable disorders, e.g., to detect gene mutations. They are also used in the diagnosis of infectious diseases (e.g., diphtheria), in forensics, and in tumor diagnostics. Specimens include DNA from nucleated blood cells or, in prenatal diagnostics, chorionic villi.

Genetic markers

Numerous loci vary dramatically in a population. Repetitive sequences of various lengths are present in several noncoding regions of the genome. These repetitive sequences differ in sequence motif length. The frequency of repetition differs in each individual. Polymorphisms in DNA form the basis, e.g., for the diagnosis of diseases and allow individuals to be identified.

Detection of gene mutations (DNA diagnostics, molecular genetic testing)

DNA diagnostics is suitable for the direct or indirect detection of a gene mutation that results in disease. It can also be used to exclude the presence of a gene mutation.

Direct detection

Various methods can be used in the direct detection of gene mutations.

Indirect detection (genetic linkage analysis)

Indirect DNA diagnostics are performed if direct detection is not possible.

  • Prerequisite: The disease occurs in several family members and the suspected locus is known.
  • Principle: analysis of genetic markers associated with the mutated gene and comparison of the patient's genotype with that of unaffected family members
  • Result: There is no direct detection of a gene mutation, but a probability of the presence of a certain mutation that causes disease (genetic risk score) can be calculated. The validity of indirect DNA diagnostics depends on the pattern of inheritance and the number of family members being investigated.

Identification of chromosomes

Karyotyping can be used to visualize chromosomes for examining chromosome numbers and for an overview of potential structural changes within a chromosome. Staining is used to visualize the special banding patterns that are characteristic for every chromosome.

  • Karyogram
    • Determines number, size, morphology, banding pattern, and arm-length ratio of chromosomes
    • Can be performed on samples from various tissues (e.g., amniotic fluid, bone marrow, placenta, blood).
    • Helpful for diagnosis of trisomies, monosomies, and sex chromosome disorders.
  • Banding pattern: transverse bands of various width and distribution, which can be induced depending on the preparation and staining technique
    • Preparation
    • Banding techniques
      • Staining with quinacrine (fluorescent bands; not used routinely in diagnostics)
      • Giemsa banding (standard banding technique, results in dark G bands with transcriptionally inactive chromatin and bright, transcriptionally active R bands)
    • Analysis: assessment of an average 10–15 metaphase chromosome pairs in 1250x magnification
    • Results

Fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization is a method used for the specific staining of DNA sequences by a fluorescence-labeled DNA or RNA probe, e.g., to stain chromosomes in karyograms, in tumor diagnosis, or to map specific genes on chromosomes in metaphase.

DNA microarray (array comparative genome hybridization, CGH)

Mainly used to simultaneously examine expression levels of multiple genes or to genotype many regions at the same time.

  • Procedure
    1. Preparation of the sample
      • A sample (m)RNA and control (m)RNA are converted to complementary DNA (cDNA) by reverse transcriptase.
      • cDNA is then labeled with fluorescent dye, one color for the sample that is being tested, another color for the control (e.g., green for the control, red for the sample being tested).
      • mRNA is removed and samples are combined.
    2. Preparation of the chip
      • Many known genetic sequences of nucleic acid (DNA or RNA) probes are arranged on a chip (e.g., glass, silicon).
      • Both patient and control DNA are applied to this chip together.
      • The chip is mounted to a scanner that can detect complementary binding of probes and sample sequences.
      • Each region of the chip stands for a known genetic sequence.
      • The higher the expression of the gene in one sample, the more intense the fluorescence → high expression of the gene in the sample being tested → e.g., intense red fluorescence
  • Uses

References:[3]

CRISPR/Cas9

CRISPR/Cas9 is a highly specific and easy to adapt gene editing system that is used for genetic engineering of cell lines to whole organisms. With CRISPR/Cas9, precise excision and addition of genes in any DNA sequence are possible. It is based on an adaptive immune response mechanism of prokaryotes to foreign gene sequences.

Adaptive prokaryotic immune response

  1. Foreign DNA is incorporated into own DNA at the CRISPR locus (acquisition).
  2. The CRISPR locus is transcribed together with foreign DNA → forms primary transcript.
  3. Primary transcript binds tracrRNA and is processed to form a crRNA-tracrRNA hybrid, which contains a foreign genetic sequence.
  4. crRNA-tracrRNA hybrid forms a complex with Cas9.
  5. Now foreign DNA that contains the complementary sequence to the one contained by the crRNA/tracrRNA/Cas9 complex can be recognized and cleaved.

CRISPR/Cas9 in gene editing

For gene editing purposes, tracrRNA and crRNA are combined into one molecule, the single synthetic guide RNA (sgRNA). There are three major variants of Cas9 editing that are used to date.

  • Wild-type Cas9
    • Cleaves dsDNA
    • Can be repaired by:
      • Nonhomologous end joining pathway → insertion or deletion of the targeted gene
      • Homology-directed repair (HDR) pathway → insertion of donor DNA → mutation in gene of interest
  • Cas9D10A
    • Cleaves only one DNA strand (nickase activity)
    • More specific because it does not activate NHEJ but only the high-fidelity HDR pathway
  • Nuclease-deficient Cas9
    • Mutations in nuclease domains prevent cleavage but not binding
    • Can be used to activate or silence genes by creating fusion proteins of Cas9 with effector proteins
  • 1. Kaplan. USMLE Step 1 Biochemistry and Medical Genetics Lecture Notes 2016. ‎Fort Lauderdale, FL: Kaplan Publishing; 2015.
  • 2. Lequin RM. Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA). Clin Chem. 2005; 51(12): pp. 2415–2418. doi: 10.1373/clinchem.2005.051532.
  • 3. Scitable Staff. Microarray. https://www.nature.com/scitable/definition/microarray-202. Updated January 1, 2018. Accessed July 19, 2018.
last updated 09/11/2018
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