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.
  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.
  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
  • 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)
  • Measures amount of antigens or antibodies
  • Confirmation of the presence of a protein in a sample (e.g. confirmatory test for antibodies)
  • Rough estimation of amount of protein in the sample

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

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 anneal to 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


Western blot (immunoblot)

Southern blot

Enzyme-linked immunosorbent assay (ELISA)


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.



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.


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.

  • Karyotyping
  • 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
      • Karyotype: evaluation of the karyogram according to the number and the structure (morphology, length, arm-ratio, pattern of banding, size) of the chromosomes
      • The total number of chromosomes is stated first followed by the type of sex chromosome present. Anomalies, if present, are noted mentioned last.

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
      • Thousands of genetic sequences of nucleic acid (DNA or RNA) probes are attached to a chip (e.g., glass, silicon).
      • Both patient and control DNA is applied to this chip and hybridizes to the probes on the chip.
      • 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




CRISPR gene editing is a technique in genetic engineering that employs the prokaryote CRISPR/Cas9 defense system directed against foreign gene sequences to modify the genomes of living organisms (e.g., adding or deleting genes in DNA sequences).

  • CRISPR (clustered regularly interspaced short palindromic repeats)
  • Cas9 (CRISPR-associated system 9)
    • Enzyme (endonuclease) that produces single or double-strand breaks at a specific nucleotide sequence guided by a site-specific RNA (targeted DNA double-strand break)
      • In prokaryotes, the guide RNA consists of crRNA and tracrRNA.
      • For laboratory use, a single guide RNA is specifically designed and synthesized.
    • The cas9 gene sequence is found adjacent to the CRISPR sequence.
  • tracrRNA (transactivating crRNA): RNA sequence that is partially complementary to crRNA; also binds to Cas9 (needed for Cas9 maturation)

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 and forms the primary transcript.
  3. The 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 sequence complementary 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) that is complementary to the DNA sequence of interest (target DNA). There are three major variants of Cas9 used in CRISPR gene editing:

  • Wild-type Cas9
  • 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

Potential applications of CRISPR/Cas9

Some of the potential applications of CRISP/Cas9 system (not currently used in clinical medicine) include:

Hemoglobin electrophoresis


A screening test that detects and quantifies the types of hemoglobins present in a sample by separating them based on their electrical charge.



  • A sample of the patient's hemoglobin is obtained by hemolysing a sample of blood using a hemolysate reagent
  • The hemoglobin sample is added to the gel electrophoresis buffer
  • An electric field is applied to the buffer that causes the different hemoglobin types to separate according to their electrical charge
  • A stain is applied to the gel to make the charged molecules visible
  • Hemoglobin is negatively charged at an alkaline pH and migrates on the gel towards the anode, forming a band
  • The degree of negative charge of the hemoglobin molecule determines the migration speed and distance from the cathode to the anode

To remember the migration speed of the different hemoglobin molecules, think: A FaSt Car can go far (A > F > S > C).

Expected findings

Please also see “Hemoglobin patterns” in thalassemia, sickle cell disease, and hemoglobin C disease.