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:
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.
- The seperated molecules are transferred from the gel onto a membrane. This transfer is called “blotting”.
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 (immunoglobulins that bind to a single, specific antigen) are used for the detection of the protein of interest in Western blot.
- 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: RNA
- 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.
- 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
- Sample: Proteins
- Principle of detection: Antibody bound to protein of interest
- Uses (examples): Confirmatory test to detect antibodies against Borrelia ( )
- Variation: Southwestern blot
- Sample: DNA (DNA restriction fragments)
- Result: double-stranded DNA; one unlabeled strand (cleaved DNA sample); one labeled strand that can be visualized when the membrane is exposed to a film.
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.
- There are two general types of ELISA tests:
- The so-called sandwich ELISA is usually performed as an indirect ELISA.
- 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.
- The specific antibody-enzyme conjugate is added to the solution.
- A substrate for the enzyme is added
- Spectrometry is used to detect the generated chromophore
- Same procedure as but
- A surface plate is coated with capture antibodies (not the patient's antibodies).
- The sample is added to the plate.
- Antigens present will be captured by capture antibodies coating the surface plate.
- Specific (labeled) antibodies for the antigen are added.
- A substrate for the enzyme is added (color, fluorescent, or electrochemical changes are due to the reaction between substrates and enzymes).
- Spectrometry, fluorescence, or electrochemical studies are performed to assess for the amount of antigens present.
- Screening for HIV antibodies (high sensitivity, low specificity)
- Testing for West Nile virus antibodies
- Detection of the following organisms
- Detection of
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).
- Complementary primers are added to DNA sample (primer pair selects the region of DNA to be amplified).
- 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.
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
- Cooling of the sample
- Elongation and amplification
- Detection of HIV (particularly when ELISA and Western blot are inconclusive)
- Forensic cases (comparison of DNA samples from suspects)
- Paternity testing
- Diagnosis of bacterial and viral infections
- Diagnosis of immunodeficiencies
- Sequencing of mutations
- Reverse transcriptase polymerase chain reaction (RT-PCR)
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.
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.
- Genetic markers (or DNA markers): individual differences in the DNA sequence of a specific region of the genome
- Three different markers are used for DNA analysis.
- short tandem repeats, STRs): repetitive sequences of several base pairs in DNA that are highly polymorphic (
- SNPs (single nucleotide polymorphism): DNA sequence variants in a population that differ by only a single base pair. They are usually caused by errors during DNA replication and are point mutations.
RFLP (restriction fragment length polymorphism): Depending on the DNA sequence of an individual, cleaving of chromosomal DNA with restriction enzymes leads to DNA fragments of variable lengths.
- The fragments are analyzed using Southern blot and detected by probes.
- Genetic fingerprint: the DNA profile of an individual, especially as determined by microsatellite analysis
Detection of gene mutations (DNA diagnostics, molecular genetic testing)
Various methods can be used in the direct detection of gene mutations.
- Prerequisite: The causal gene for the (suspected) disease is known.
- Amplification of a specific region of the affected gene using PCR
- Detection of a mutation, e.g., through sequencing or cleavage of DNA fragments using restriction enzymes
- Restriction enzymes (restriction endonucleases)
- Analysis via gel electrophoresis: Detection is based on the altered mobility of the DNA fragments of mutated and normal alleles in gel electrophoresis.
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.
Banding pattern: transverse bands of various width and distribution, which can be induced depending on the preparation and staining technique
- 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
- 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 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.
- Denaturation of DNA in the prepared chromosomes
- Hybridization of the DNA with a single-stranded, fluorescence-labeled DNA probe that is complementary to a specific DNA sequence
Analysis of the chromosome set in fluorescence microscopy
- A fluorescent signal indicates the site of the bound probe.
- Microdeletion: the deleted region does not exhibit fluorescence, compared to the region present in the same locus of the other copy of the chromosome.
- Translocation: fluorescence signal from one chromosome is present in a different chromosome
- Duplication: extra copies of chromosomes that exhibit fluorescence in addition to the normal ones
- Uses: detection of microdeletions (2 - 3 × 106 base pairs) such as in DiGeorge syndrome; evidence of a Philadelphia chromosome translocation t(9;22).
DNA microarray (array comparative genome hybridization, CGH)
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.
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
- Preparation of the sample
- Helpful for detection of copy number variations (CNVs), single nucleotide polymorphisms (SNPs) that are used for genotyping, forensic science, cancer mutations, genetic linkage analysis, and genetic testing.
- Sequencing: determination of the exact sequence of base pairs of a gene; e.g., to demonstrate an unknown mutation on a disease-causing gene
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.
- CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)
Cas9 (CRISPR-associated system 9)
- Enzyme (endonuclease) that cleaves DNA double strands at a specific nucleotide sequence guided by a site-specific RNA (“targeted DNA double-strand break”)
- The cas9 gene sequence is found adjacent to the CRISPR sequence.
- tracrRNA (transactivating crRNA): a separate RNA sequence that is partially complementary to crRNA → also binds to Cas9 (needed for Cas9 maturation)
Adaptive prokaryotic immune response
- Foreign DNA is incorporated into own DNA at the CRISPR locus (acquisition).
- The CRISPR locus is transcribed together with foreign DNA → forms primary transcript.
- Primary transcript binds tracrRNA and is processed to form a crRNA-tracrRNA hybrid, which contains a foreign genetic sequence.
- crRNA-tracrRNA hybrid forms a complex with Cas9.
- 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
- Wild-type Cas9
- Nuclease-deficient Cas9
- Potential applications of CRISPR/Cas9 system (not currently used in clinical medicine)
A screening test that detects and quantifies the types of hemoglobins present in a sample by separating them based on their electrical charge.
- Diagnostic work-up of hemolytic anemias
- Screening for or evaluation of hemoglobinopathies in high-risk individuals (e.g., positive family history)
- Evaluation of abnormal erythrocytes on peripheral blood smear
- 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
- HbA migrates the fastest and therefore the greatest distance, followed by Hb F, Hb S, and Hb A2, and Hb C (A > F > S > A2 and C)
- Hemoglobin C migrates the least because a missense mutation replaces the negatively charged glutamic acid with the positively charged lysine.
- Hemoglobin S migrates less than HbA because of the replacement of the negatively charged glutamic acid with the neutral valine.
To remember the migration speed of the different hemoglobin molecules, think: A FaSt Car can go far (A > F > S > C).
- Normal (adult): wide HbA band (AA)
- Normal (fetal: ): HbA and widened HbF band (AF)
- Beta thalassemia minor (trait): narrowed HbA band and widened HbA2 band
- Beta thalassemia major: no HbA band; widened HbA2 and HbF bands
- Sickle cell trait: HbA and HbS bands (AS)
- Sickle cell anemia: no HbA band; wide HbS band (SS)
- HbC trait: HbA and HbC bands (AC)
- HbC disease: no HbA band; wide HbC band (CC)
- HbSC disease: no HbA band; HbS and HbC bands (SC)
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