molecular diagnostics by lela buckingham

Initially, these methods did not provide the efficient recovery of clean DNA achieved with phenol extraction however, newer methods have proven to produce high quality DNA preparations in good yields. Molecular Diagnostics DNAInorganic Isolation Methods “salting out” Have been developed to avoid use of toxic reagents such as phenol.

DNA pellet is dissolved in rehydration buffer, usually 10 mM Tris, 1 mM EDTA (TE), or water.

Excess salt is removed by rinsing the pellet in 70% ethanol, centrifuging and discarding the ethanol supernatant.

Upon centrifugation, DNA forms a solid precipitate.

DNA is precipitated using ethanol (at 2:1 ratio) or isopropanol (at 1:1 ratio) in a high concentration of salt (ammonium, potassium or sodium acetate, or lithium or sodium chloride).

The upper phase containing the DNA is collected.

Collects a white precipitate of amphiphilic components, as well as cell debris.

Middle interface between the two layers:.

The hydrophilic layer (aqueous phase) on top dissolves DNA.

Dissolves lipids and other hydrophobic components.

When added to the hydrophilic cleared cell lysate, a biphasic emulsion forms.

Molecular Diagnostics DNAOrganic Isolation Methods Further purification of DNA from contaminating proteins, lipids, carbohydrates, and cell debris using a combination of high salt, low pH, and an organic mixture of phenol and chloroform. Amplification methods will work with this type of DNA isolation.

The advantage of these types of extraction is their speed and simplicity.

DNA extracted with NaOH or boiling procedures is denatured (single-stranded) and may not be suitable for methods such as restriction enzyme analysis that require double-stranded DNA.Gentler enzymatic methods are less likely to damage chromosomal DNA and thus are preferred for methods involving larger chromosomal targets as opposed to plasmid DNA.Boiling in 8% sucrose, 8% Triton X-100 detergent, Tris buffer, and EDTA after lysozyme treatment releases DNA that can be immediately precipitated with alcohol.Treatment with detergent (1% sodium dodecyl sulfate) and strong base (0.2 M NaOH) in the presence of Tris base, ethylenediaminetetraacetic acid (EDTA), and glucose can also break bacterial cell walls.Alternatively, cell walls can be broken mechanically by grinding or by vigorously mixing with glass beads.By several enzyme products, e.g., lyzozyme or zymolyase, that digest cell wall polymers.Molecular Diagnostics Bacteria and fungi having tough cell walls must be broken to allow the release of nucleic acid After xylene treatment, the tissue is usually rehydrated by soaking it in decreasing concentrations of ethanol.Fixed embedded tissue has to be deparaffinized by soaking in xylene (a mixture of three isomers of dimethylbenzene).Whole tissue samples are disrupted by grinding the frozen tissue in liquid nitrogen, homogenizing the tissue, or simply mincing the tissue using a scalpel.Molecular Diagnostics Fresh or frozen tissue samples must be dissociated The WBCs are then pelleted by centrifugation, leaving the empty RBC membranes (ghosts) and hemoglobin, respectively, in suspension and solution.Hypotonic buffer or water will result in the lysis of the RBCs before the WBCs.Takes advantage of the differences in the osmotic fragility of RBCs and WBCs.

Isolation of WBCs by differential lysis:.Remove from the tube and wash by at least 2 rounds of resuspension and centrifugation in saline.Centrifuge: Mononuclear WBCs (the desired cells for isolation of nucleic acid) settle into a layer.Overlay with Ficoll (highly branched sucrose polymer that does not penetrate biological membranes).Isolation of WBCs by differential density gradient centrifugation:.Molecular Diagnostics Whole blood may have to be pretreated to make nucleated cells available Nucleic Acid Extraction Methods Molecular Diagnostics Genotyping and identification of date palmĭon’t forget to read “Advanced Concepts” In the textbook MOLECULAR DIAGNOSTICS: Fundamentals, Methods, & Clinical Applications Molecular Diagnostics.Diagnostic and Prognostic Significance of the Methylation Status in cancers.Determination of genetic factors for human obesity.Detection of point mutations in thrombophilia factors.Identification, determination of virulence factors and determination of drug resistance in H.

Typing and subtyping of influenza viruses.

Quantitative determination of Hepatitis viral load.

Applications in diagnosis of viral and bacterial infections:.

Applications of molecular testing in diagnosis.

Chromosomal Structure and Chromosomal Mutations.

Analysis and Characterization of Nucleic Acids and Proteins.

Resolution and Detection of Nucleic Acids.

MOLECULAR DIAGNOSTICS: Fundamentals, Methods, & Clinical Applications, 2007, F.

Molecular Diagnostics Molecular Diagnostics

Molecular diagnostics by lela buckingham