Traditional gel electrophoresis utilizes an electric current, a buffer, and a porous matrice of agarose or polyacrylamide for the separation of nucleic acid molecules according to size. As the electrical current is applied to the system, the negatively charged nucleic acids will migrate toward the positive pole or anode. Electrophoresis may utilize a horizontal or vertical gel apparatus or a small tube or capillary system. Capillary electrophoresis utilizes a thin glass silica capillary tube for faster separation and detection using fluorescent detection. Agarose is a polysaccharide polymer that is extracted from seaweed. It is relatively inexpensive and easy to use. Poly acrylamide is typically a mixture of acrylamide and a cross-linking methylene bisacrylamide. Polyacrylamide is a more porous or highly cross-linked gel that provides for a higher resolution of smaller fragments and single stranded molecules. Despite the higher resolving power of acrylamide gels, it is important to note that in the powder form and unpolymerized form, acrylamide is neurotoxic, and proper safety precautions should be used during handling.

In addition to varying systems and matrices, different buffers may be used for the separation of nucleic acids. The two most common buffering systems include Trias acetate or Tris borate buffers. Tris borate EDTA (TBE, 0.089 M Tris-base, 0.089 boric acid, 0.0020 M EDTA) has a greater buffering capacity. However, TBE has a tendency to precipitate during storage and generates heat during electrophoresis. Excessive heating during electrophoresis can result in distorted patterns and make detection or interpretation of migration patterns difficult. Tris acetate EDTA (TAE, 0.04 M Tris-base, 0.005 M sodium acetate, 0.002 M EDTA) provides for faster migration or separation during electrophoresis. Denaturing agents such as detergents, formamide, or urea may be added to the buffers that break the hydrogen bonds between complementary sequences on DNA or RNA molecules that may alter migration patterns.

Pyrosequencing

Traditional nucleic acid sequencing is based on chain termination and the addition of a labeled nucleotide (TTP, GTP, ATP, CTP, or UTP) that is then detected using a radiolabeled or fluorescent tag. Pyrosequencing is a newer method that incorporates a luminescent signal (generation of a pyrophosphate) when nucleotides are added to the growing nucleic acid strand. The reaction incorporates a sequencing primer that hybridizes to the single-stranded target. The hybrids are incubated with DNA polymerase, ATP sulfurylase, luciferase, and apyrase along with the substrates adenosine-5′-phosphosulfate and luciferin. A single dNTP (deoxynucleotide triphospate) is added to the reaction. As the polymerase extends the target from the primer, the dNTP is incorporated, releasing a pyrophosphate (PPi). The ATP sulfurylase then converts the PPi to ATP, which drives the conversion of luciferin to oxyluciferin, generating light. The amount of light generated is proportional to the amount of the specific nucleotide incorporated, generating a report or pyrogram. The Apyrase degrades the ATP and unincorporated dNTPs, turning off the light and regenerating the reaction mixture. The next dNTP is added, repeating the process for each subsequent nucleotide. Pyrosequencing is useful for identifying drug- resistant mutations and identification of viral, bacterial, or fungal nucleic acids.

High-Density DNA Probes

 An alternative to gel-based sequencing has been the introduction of the high-density oligonucleotide probe array. This technology was developed recently by Affymetrix (Santa Clara, California). The method relies on the hybridization of a fluorescent-labeled nucleic acid target to large sets of oligonucleotides synthesized at precise locations on a miniaturized glass substrate that may include glass or “chip” or siliconized wafer. The hybridization pattern of the probe to the various oligonucleotides is then used to gain primary structure information about the target (Figure 1). Hybridization high-density microarrays in combination with sequence-independent amplification (PCR) have also been used to identify pathogens. This technology has been applied to a broad range of nucleic acid sequence analysis problems, including pathogen identification and classification, polymorphism detection, and drug-resistant mutations for viruses (e.g., HIV) and bacteria.

Fig1.  Overview of high-density DNA probes. High-density  oligonucleotide arrays are created using light-directed chemical syn thesis that combines photolithography and solid-phase chemical  synthesis. Because of this sophisticated process, more than 500 to  as many as 1 million different oligonucleotide probes may be formed  on a chip; an array is shown in A. Nucleic acid is extracted from a  sample and then hybridized within seconds to the probe array in a  GeneChip Fluidics Station. The hybridized array (B) is scanned using  a laser confocal fluorescent microscope that looks at each site (i.e.,  probe) on the chip, and the intensity of hybridization is analyzed  using imaging processing software. 

Low- to Moderate-Density Arrays

Improved technology in molecular diagnostics has resulted in the development of low- to moderate-density microarray platforms that are less expensive than high density arrays. This has allowed many laboratories to incorporate this new and powerful technology into the daily operations of the diagnostic microbiology laboratory. These microarrays utilize layered film, gold-plated electrodes, and electrochemical detection or gold nanoparticles for the detection of target sequences. There are currently three FDA-approved platforms available in the United States: the INFINITI analyzer (Autoge nomics, Vista, CA), the eSensor XT-8 system, (GenMark Diagnostics, Carlsbad, CA), and the Verigene system (Nanosphere Inc., Northbrook, IL). These instruments are closed-system, random access, completely automated systems, making the detection of nucleic acids relatively simple and free from the hazards of contamination by other circulating nucleic acids or amplification products.

Enzymatic Digestion and Electrophoresis of Nucleic Acids

 Enzymatic digestion and electrophoresis of DNA fragments are not as specific as sequencing or specific amplification assays in identifying and characterizing microorganisms. However, enzyme digestion-electrophoresis procedures still provide valuable information for the diagnosis and control of infectious diseases.

Enzymatic digestion of DNA is accomplished using any of a number of enzymes known as restriction endo nucleases. Each specific endonuclease recognizes a specific nucleotide sequence (usually 4 to 8 nucleotides in length), known as the enzyme’s recognition, or restriction, site. Restriction sites are often palindromic sequences; in other words, the two strands have the same sequence, which run antiparallel to one another. Once the recognition site has been located, the enzyme catalyzes the digestion of the nucleic acid strand at that site, causing a break, or cut, in the nucleic acid strand (Figure 2).

Fig2. DNA enzymatic digestion and gel electrophoresis to  separate DNA fragments resulting from the digestion. An example  of a nucleic acid recognition site and enzymatic cut produced by  EcoR1, a commonly used endonuclease, is shown in the inset. 

The number and size of fragments produced by enzymatic digestion depend on the length of nucleic acid being digested (the longer the strand, the greater the likelihood of more recognition sites and thus more fragments), the nucleotide sequence of the strand being digested, and the particular enzyme used for digestion. For example, enzymatic digestion of a bacterial plasmid whose nucleotide sequence provides several recognition sites for endonuclease A, but only rare sites for endo nuclease B, will produce more fragments with endonuclease A. Additionally, the size of the fragments produced will depend on the number of nucleotides between each of endonuclease A’s recognition sites present on the nucleic acid being digested.

The DNA used for digestion is obtained by various methods. A target sequence may be obtained by amplification via PCR, in which case the length of the DNA to be digested is relatively short (e.g., 50 to 1000 bases). Alternatively, specific procedures may be used to cultivate the organism of interest to large numbers (e.g., 1010 bacterial cells) from which plasmid DNA, chromosomal DNA, or total cellular DNA may be isolated and purified for endonuclease digestion.

After digestion, fragments are subjected to agarose gel electrophoresis, which allows them to be separated according to their size differences as previously described for Southern hybridization. During electrophoresis all nucleic acid fragments of the same size comigrate as a single band. For many digestions, electrophoresis results in the separation of several different fragment sizes (Figure 3). The nucleic acid bands in the agarose gel are stained with the fluorescent dye ethidium bromide, which allows them to be visualized on exposure to UV light. Stained gels are photographed for a permanent record (see Figure 3; also Figures 4 and 5).

Fig3. Restriction fragment length polymorphisms of  vancomycin-resistant Enterococcus faecalis isolates in Lanes A  through G as determined by pulsed-field gel electrophoresis. All  isolates appear to be the same strain. 

Fig4. Although antimicrobial susceptibility profiles indicated  that several methicillin-resistant S. aureus isolates were the same  strain, restriction fragment length polymorphism analysis using  pulsed-field gel electrophoresis (Lanes A through F) demonstrates  that only isolates B and C were the same. 

Fig5. Restriction patterns generated by pulsed-field gel electrophoresis for two Streptococcus pneumoniae isolates, one that  was susceptible to penicillin (Lane B) and one that was resistant  (Lane C), from the same patient. Restriction fragment length poly morphism analysis indicates that the patient was infected with different strains. Molecular-size markers are shown in Lane A. 

One variation of this method, known as ribotyping, involves enzymatic digestion of chromosomal DNA followed by Southern hybridization using probes for genes that encode ribosomal RNA. Because all bacteria contain ribosomal genes, a hybridization pattern will be obtained with almost any isolate, but the pattern will vary depending on the arrangement of genes in a particular strain’s genome.

Regardless of the method, the process by which enzyme digestion patterns are analyzed is referred to as restriction enzyme analysis (REA). The patterns obtained after gel electrophoresis are referred to as restriction patterns, and differences between microorganism restriction patterns are known as restriction fragment length polymorphisms (RFLPs). Because RFLPs reflect differences or similarities in nucleotide sequences, REA methods can be used for organism identification and for establishing strain relatedness within the same species (see Figures 3 to 5).

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مواضيع ذات صلة

Analysing Genes and Gene Expression

Screening Gene Libraries

Hybridisation and Gene Probes

Cloning Vectors: Vectors Used in Eukaryotes

Addition of Telomeric Sequences by Telomerase Prevents Shortening of Chromosomes

Centromere Sequences Vary Greatly in Length and Complexity

Interphase Polytene Chromosomes Arise by DNA Amplification

Cloning Vectors : Cosmid-Based Vectors

Cloning Vectors : Phagemid-Based Vectors

Cloning Vectors : Virus-Based Vectors

Cloning Vectors : Plasmids

The TATA Box, Initiators, and CpG Islands Function as Promoters in Eukaryotic DNA