Many of the revolutionary changes that have occurred in research in the biological sciences, particularly the Human Genome Project, can be directly attributed to the ability to manipulate DNA in defined ways. Molecular genetic testing focuses on the examination of nucleic acids (DNA or RNA) by special techniques to determine whether a specific nucleotide base sequence is present.

The applications of nucleic acid testing have expanded, despite higher costs associated with testing, in various areas of the clinical laboratory. These include genetic testing, hematopathology diagnosis and monitoring, and identification of infectious agents. Molecular testing has the following advantages:

 • Faster turnaround time

• Smaller required sample volumes

• Increased specificity and sensitivity

Conventional Analysis

Detection of DNA products by PCR assay can be convention ally analyzed using agarose gel electrophoresis after ethidium bromide staining. This technique is simply an extra step after a PCR assay has been run. DNA and other biomolecules can be separated based on charge, size, and shape. DNA has a net negative charge and will migrate toward the anode (positive pole). PCR products are loaded into an agarose gel and electrophoresed. Ethidium bromide is a dye that intercalates into nucleic acids and will fluoresce with an orange color under ultraviolet (UV) irradiation. An image analyzer uses UV light to capture computer images of the PCR products.

Other Techniques

 Other techniques are used to enhance the sensitivity and specificity of amplification techniques. Probe-based DNA detection systems have the advantage of providing sequence specificity and lower detection limits. Other techniques include the hybridization protection assay, DNA EIA, automated DNA sequencing technology, single-strand conformational poly morphism, and restriction fragment length polymorphism (RFLP) analysis. The selection of one technique over another is often based on factors such as sensitivity and specificity pro files, cost, turnaround time, and local experience.

DNA Sequencing

 DNA sequencing is considered to be the gold standard to which other molecular methods are compared. DNA sequencing dis plays the exact nucleotide or base sequence of a fragment of DNA that is targeted. The Sanger method, which uses a series of enzymatic reactions to produce segments of DNA complementary to the DNA being sequenced, is the most frequently used method for DNA sequencing. Automated sequencing techniques use primers with four different fluorescent labels.

1. The first step in sequencing a target is usually to amplify it by cloning or in vitro amplification, usually PCR. Once the amplified DNA is purified from the clinical specimen (the target DNA), it is heat-denatured to separate the double stranded DNA (dsDNA) into single strands (ssDNA).

2. The second step involves adding primers to the ssDNA. Primers are short synthetic segments of ssDNA that contain a nucleotide sequence complementary to a short strand of target DNA. The patient’s DNA serves as a template to copy. DNA polymerase catalyzes the addition of the appropriate nucleotides to the preexisting primer. DNA synthesis is terminated when the deoxynucleotide is incorporated into a growing DNA chain.

Branched DNA

 Branched DNA (bDNA) is another quantitative test that uses signal amplification instead of target amplification. Target DNA or RNA is hybridized at different sites by two types of probes. Branched DNA assays are used to measure the viral load of hepatitis B virus (HBV), HCV, HIV-1, CMV, and microbial organisms (e.g., Trypanosoma brucei).

T he Versant HIV-1 RNA 3.0 assay (bDNA; Bayer, Berkeley, Calif), uses bDNA technology. It is the only viral load assay specifically designed to target multiple sequences of the HIV-1 genome with more than 80 nucleic acid probes.

Hybridization Techniques

 Many forms of probe hybridization assays involve the complementary pairing of a probe with a DNA or RNA strand derived from the patient’s specimen. The common feature of probe hybridization assays is the use of a labeled nucleic acid probe to examine a specimen for a specific, homologous DNA or RNA sequence. Clinical probes are usually labeled with nonradioisotopic molecules such as digoxigenin, alkaline phosphatase, bio tin, or a fluorescent compound. The detection systems are conjugate-dependent and include chemiluminescent, fluorescent, and calorimetric methodologies.

Liquid-Phase Hybridization

In the liquid-phase hybridization (LPH) assay, the target nucleic acid and labeled probe interact in solution. Specific homologous hybrids are subsequently separated from the remaining nucleic acid component and the hybrids are identified by an appropriate detection system.

Dot Blot and Reverse Dot Blot

These hybridization methods are used in the clinical laboratory for the detection of disorders in which the DNA sequence of the mutated region has been identified (e.g., sickle cell anemia, cystic fibrosis). These techniques are capable of distinguishing the homozygous or heterozygous state of a mutation.

Dot Blot. The dot blot hybridization method detects single-base mutations using allele-specific oligonucleotides (ASOs). Unlike other assays, dot blot does not require enzyme digestion or electrophoretic separation of DNA fragments. The procedure uses labeled oligonucleotide probes of about 15 to 19 bp. DNA is amplified in the region of a known mutation, denatured, and applied to separate areas of a membrane or filter. A probe designed to detect a normal DNA sequence is added to one area; a second probe for the detection of a sequence with the single-base mutation is applied to a second area. Ideally, only the labeled probe whose base sequences perfectly match those of the patient will hybridize.

Reverse Dot Blot. In this variation of the dot blot procedure, the ASO probes are bound to a filter and denatured DNA from the patient is added to the immobilized ASO. Hybridization occurs only if the patient’s DNA contains base sequences that are 100% complementary to those of the probe. A common variation of the reverse dot blot procedure is to bind oligonucleotide probes of a slightly longer length than usual to a 96-well microtiter plate. Biotin is used to label copies of the target sequence. The labeled copies are hybridized in the wells to the bound probes and detected using avidin conjugated to horseradish peroxidase. Subsequent addition of substrate pro duces a colored reaction that can be read photometrically.

Blotting Protocols

 The Southern blot and Northern blot techniques are used to detect DNA and RNA, respectively. These procedures share the following steps:

 1. Electrophoretic separation of the patient’s nucleic acid

2. Transfer of nucleic acid fragments to a solid support (e.g., nitrocellulose)

3. Hybridization with a labeled probe of known nucleic acid sequence

4. Autoradiographic or colorimetric detection of the bands created by the probe–nuclei acid hybrid

Southern Blot. Specimen DNA is denatured and treated with restriction enzymes to create DNA fragments; then the ssDNA fragments are separated by electrophoresis (Figure 1). The electrophoretically separated fragments are then blotted to a nitrocellulose membrane, retaining their electrophoretic position and hybridized with radiolabeled single-stranded DNA fragments with sequences complementary to those being sought. The resulting dsDNA bearing the radiolabel, if present, is then detected by radiography.

Fig1. Identification by Southern blot hybridization of DNA  fragment containing gene X. DNA was digested with restriction endo nuclease, and resulting fragments were fractionated according to  size by electrophoresis in agarose gel. DNA fragments in gel were  denatured and blotted to nitrocellulose filter as a result of flow of buf fer through gel and nitrocellulose filter to dry paper towels. Subse quent hybridization of DNA on filter to 32P-labeled gene X probe and  autoradiography revealed single DNA fragment containing gene X.  (Reprinted with permission from LeGrys V, Leinbach SS, Silverman L: CRC Crit Rev Clin Lab Sci 25:255, 1987. Copyright CRC Press, Boca Raton, FL.)

The Southern blot procedure has clinical diagnostic applications for disorders associated with significant changes in DNA, a deletion or insertion of at least 50 to 100 bp (e.g., fragile X syndrome), and determination of clonality in lymphomas of T or B cell origin. If a single-base mutation changes an enzyme restriction site on the DNA, resulting in an altered band or fragment size, the Southern blot procedure can detect these changes in DNA sequences, referred to as RFLPs. Single-base mutations that can be determined by the Southern blot technique include sickle cell anemia and hemophilia A.

Northern Blot. mRNA from the specimen is separated by electrophoresis and blotted to a specially modified paper support; this results in covalent fixing of the mRNA in the electrophoretic positions. Radiolabeled, ssDNA fragments complementary to the specific mRNA being sought are then hybridized to the bound mRNA. If the specific mRNA is present, its radioactivity is detected by autoradiography.

The derivation of this technique from the Southern blot technique used for DNA detection has led to the common usage of the term Northern blot for the detection of specific mRNA. However, the Northern blot technique is not routinely used in clinical molecular diagnostics.

Western Blot. Compared with the Southern blot technique, which separates and identifies RNA fragments and proteins, and the Northern blot technique, which concentrates on isolating mRNA, in the Western blot technique proteins are separated electrophoretically, transferred to membranes, and identified through the use of labeled antibodies specific for the protein of interest (Fig. 2).

Fig2. Western blot immunoassay. (Adapted from Forbes BA, Sahm DF, Weissfeld AS: Bailey & Scott’s diagnostic microbiology, ed 12, St Louis, 2007, Mosby.)

The Western blot technique detects antibodies to specific epitopes of antigen subspecies. Electrophoresis of antigenic material results in the separation of the antigen components by molecular weight (MW). Blotting the separated antigen to nitrocellulose, retaining the electrophoretic position, and causing it to react with patient specimen will result in the binding of specific antibodies, if present, to each antigenic band. Electrophoresis of known MW standards allows for the determination of the MW of each antigenic band to which antibodies may be produced. These antibodies are then detected using EIA reactions that characterize antibody specificity.

The Western blot technique is often used to confirm the specificity of antibodies detected by enzyme-linked immunosorbent assay (ELISA) screening procedures.

Microarrays

 Microarray (DNA chip) technology has helped accelerate genetic analysis, just as microprocessors accelerated computation (Fig.3). Microarrays are basically the product of bonding or direct synthesis of numerous specific DNA probes on a stationary, often silicon-based support. The chip may be tailored to particular disease processes. The technique is easily performed and readily automated.

Fig3. Affymetrix GeneChip probe array. (Courtesy Affymetrix, Santa Clara, Calif.)

Microarrays are miniature gene fragments attached to glass chips. These chips are used to examine the gene activity of thousands or tens of thousands of gene fragments and to identify genetic mutations using a hybridization reaction between the sequences on the microarray and a fluorescent sample. After hybridization, the chips are scanned with high-speed fluorescent detectors and the intensity of each spot is quantitated (Fig. 4).

Fig4. Overview of eukaryotic target labeling for GeneChip expression arrays. (Courtesy Affymetrix, Santa Clara, Calif.)

The identity and amount of each sequence are revealed by the location and intensity of fluorescence displayed by each spot. Computers are used to analyze the data (Fig.5).

Fig5. Data from an experiment showing the expression of thousands of genes on a single GeneChip probe array. (Courtesy Affymetrix, Santa Clara, Calif.)

The applications of microarrays in clinical medicine include the analysis of gene expression in malignancies (e.g., mutations in the breast cancer 1 gene [BRCA-1], mutations of the p53 tumor suppressor gene, genetic disease testing, viral resistance mutation detection).

The Human Genome GeneChip set (HG-U133 Set; Affymetrix, Santa Clara, Calif), consisting of two GeneChip arrays, contains almost 45,000 probe sets representing more than 39,000 transcripts derived from approximately 33,000 well-substantiated human genes. The sequence clusters were created from the UniGene database and then refined by analysis and comparison with a number of other publicly available databases (e.g., Washington University EST trace repository and University of California, Santa Cruz, Golden Path human genome database).

The HG-U133A array includes representation of the Ref Seq database sequences and probe sets related to sequences previously represented on the Human Genome U95Av2 array. The HG-U133B array contains primarily probe sets representing expressed sequence tag (EST) clusters. The applications of this array include defining tissue and cell type–specific gene expression and investigating cellular and tissue responses to the environment (e.g., heat shock, interactions with other cells, exposure to chemical compounds, growth factors, or other signaling molecules). In addition, this array helps elucidate human cell differentiation by the following: (1) determining which transcripts are increased or decreased during distinct stages in cellular differentiation; and (2) detecting which genes are uniquely expressed during different stages of tumorigenesis.

Another genomic microarray, GenoSensor (Tempe, Ariz), enables researchers to screen for abnormal gene amplifications and deletions with the sensitivity to detect single-gene copy change in a variety of specimens. The GenoSensor system simultaneously screens for gene copy number changes in 287 targets spotted in triplicate. This permits the screening of proto-oncogenes, tumor suppressor genes, microdeletion syndrome, gene regions, and subtelomeric regions.

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الفلفل الأحمر.. سعرات حرارية منخفضة وفوائد صحية عديدة

8 أعراض إذا شعرت بها.. اذهب للطوارئ فورا !

تعرف على أكثر 8 أماكن اتساخا في المنزل

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مواضيع ذات صلة

Future Directions of Molecular Diagnostic Testing

Next Generation Sequencing Technology

platelet aggregation test (Ristocetin test)

plasminogen activator inhibitor 1 (PAI-1)

phospholipase A2 receptor antibodies (anti-pla2r)

phosphate (PO4 , Phosphorus [P])

pheochromocytoma suppression and provocative testing (Clonidine suppression test [CST], Glucagon stimulation test)

parvovirus B19 antibody

Applications of Molecular Biology to Erythrocyte Immunohematology

Direct Antiglobulin Test (Direct Coombs Test)

Indirect Antiglobulin Test (Indirect Coombs Test)

Partial thromboplastin time, activated (APTT, Partial thromboplastin time [PTT])