Wednesday, August 17, 2011

Molecular Techniques in Diagnosis of Infectious Diseases

Molecular microbiology is the branch of microbiology devoted to the study of the molecular principles of the physiological processes involved in the life cycle of prokaryotic and eukaryotic microorganisms such as bacteria, viruses, unicellular algae, fungi and protozoa. This includes gene expression and regulation, genetic transfer, the synthesis of macromolecules, sub-cellular organization, cell to cell communication and molecular aspects of pathogenicity and virulence.Molecular microbiology is primarily involved in the interactions between the various cell systems of microorganisms including the interrelationship of DNA, RNA , protein biosynthesis and the manner in which these interactions are regulated ( Thiel , 2007).

Techniques of molecular biology:

*Expression cloning :

One of the most basic techniques of molecular biology to study protein function is expression cloning. In this technique DNA coding for a protein of interest is cloned (using PCR and/or restriction enzymes) into a plasmid (known as an expression vector). This plasmid have special elements to drive production of the protein of interest (Diaz,2008).

This plasmid can be inserted into either bacterial or animal cells. Introducing DNA into animal cells by physical or chemical means is called transfection. Several different transfection techniques are available such as calcium phosphate ,electroporation, microinjection and liposome transfection.Introducing DNA into bacterial cells can be done by transformation via uptake of naked DNA, conjugation via cell-cell contact or by transduction via viral vector .Two types of vectors are most commonly used: E. coli plasmid vectors and bacteriophage λ vectors. Plasmid vectors replicate along with their host cells, while λ vectors replicate as lytic viruses killing the host cell and packaging the DNA into virions. In either case DNA coding for a protein of interest is now inside a
cell and the protein can now be expressed (Fratamico & Bayles , 2005).

*Polymerase chain reaction (PCR) :

PCR is an extremely versatile technique for copying DNA. In brief, PCR allows a single DNA sequence to be copied millions of times .For example PCR can be used to introduce restriction enzyme sites or to mutate (change) particular bases of DNA, the latter is a method referred to as "Quick change". PCR can also be used to determine whether a particular DNA fragment is found in a cDNA library. PCR has many variations like

reverse transcription PCR (RT-PCR) for amplification of RNA and more recently real-time PCR (QPCR) which allow for quantitative measurement of DNA or RNA molecules ( Logan et al ., 2009).

*Gel electrophoresis :

Gel electrophoresis is one of the principal tools of molecular biology. The basic principle is that DNA, RNA and proteins can all be separated by means of an electric field. In agarose gel electrophoresis DNA and RNA can be separated on the basis of size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). Shorter molecules move faster and migrate farther than longer ones. Proteins can be separated on the basis of size by using an sodium dodecyl sulfate SDS-PAGE (polyacrylamide gel electrophoresis) gel ( Mackay , 2007).

*Southern blotting :

Is named after its inventor biologist Edwin Southern.The Southern blot is a method for detections of a specific DNA sequence within a DNA sample. DNA samples before or after restriction enzyme digestion are separated by gel electrophoresis and then transferred to a membrane by blotting via capillary action. The membrane is then exposed to a labeled DNA probe that has a complement base sequence to the sequence on the DNA of interest. Most original protocols used radioactive labels, however non-radioactive alternatives are now available (Fratamico & Bayles , 2005).

*Northern blotting :

The northern blot is a technique used in molecular biology research to study gene expression. Northern blotting involves the use of electrophoresis to separate RNA samples by size, and detection with a hybridization probe complementary to part of or the entire target sequence. It takes its name from its similarity to the Southern blot technique .The major difference is that RNA, rather than DNA, is analyzed in the northern blot. Both techniques use electrophoresis and detection with a hybridization probe . The northern blot is used to study the expression patterns of specific type of RNA molecule. The procedure is also used to study when and how much gene expression is occurring by measuring how much RNA present in different samples ( Roy , 2008).

*Western blotting :

The western blot (immunoblot) is an analytical technique
used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or nylon ) where they are probed (detected) using antibodies specific to the target protein ( Mackay , 2007).

*Arrays :

DNA microarray is a multiplex technology used in molecular biology and in medicine. It consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides, called features, each containing specific DNA sequence. This can be a short section of a gene or other DNA elements that are used as probes to hybridize a cDNA or cRNA sample (called target) under high-stringency conditions. DNA microarrays can

be used to measure changes in expression levels, to detect single nucleotide polymorphisms (SNPs) , in genotyping or in resequencing mutant genomes. Microarrays also differ in fabrication, workings, accuracy, efficiency, and cost (Kostrzynska & Bachand , 2006).

Arrays can also be made with molecules other than DNA. For example an antibody array can be used to determine which proteins or bacteria are present in a blood sample (Fratamico & Bayles 2005).

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