Lab. 1 Genetic materials.pptxddfdsffefsdf

Naegleria fowleri
Pathogenesis and viruleince factors
The portal of entry into the CNS is the olfactory neuroepithelium. It is believed that the sustentacular cells lining the olfactory neuroepithelium phagocytose the amoebae that enter the nasal passages of the victims while indulging in aquatic activities. The amoebic trophozoites pass through the sieve-like cribriform plate and penetrate into the subarachnoid space and continue on to the brain parenchyma
Incubation take 5-7 days
• human mucoepithelial cells recognize N. fowleri trophozoites through TLR2/TLR4, and this further leads to pro-inflammatory cytokine production
• Extravascular neutrophil encounter with amoeba could elicit a series of responses, including reactive oxygen or nitrogen species (ROS, NOS) production, and neutrophils extracellular trap (NET) formation within the olfactory tissues.
Many virulence factors involved in pathogenesis as a following :
1-amoebostomes : sucker like structure which nipple a way in tissue
2-naegleriapore proteins (A,B) are perforin like proteins which involved in destruction and lysis of cells
3- phospholipase A ,B destruction of cell membrane
4- elastase and neuroaminidase involved in cell and extracellular destruction
5-cytopathic proteins trigger apoptosis
6-CD59 ( resist complement and prevent pore formation )
7-G-protien cr GPCR homology with maCHr1 which recive acetylcholine ant recruit naegleria through olfactory nerve
8-eveade immune response ( complement ) by express surface proteins and substances or by shedding MAC by vesicles

Acannthamoeba pathogenesis :
A-contact dependent factors
1-adherenc : by mannose binding protein and laminin binding protein
2-induce apoptosis through phosphatidylinositol 3 kinase wich induce bac and bax (proapoptosis) mediators which activate cytochrome c ( damage mitochondrial membrane ) leading to cell death
3-phagocytosis ( has the ability to phagocytosing bacteria debris fungi )
b-contact Independents factors
1-produce proteinases
( serine protease – destroy IgA and IgG
phospholipase – damage cell membrane
nuroaminidase targeting sialic acid in cornea epithelial and alter phospholipid in encephalitis
2-increase permeability of human brain micro vascular endothelial cells (HBMEC)
3- resist harsh condition and chemotherapy
4-masking their surface ( make coat from host protein ) to evading from immune response
5- produce superoxide dismutase
- Iron superoxide dismutase
- Copper-zinc oxide dismutase

6-display plasminogen activator activity by cleave plasminogen to plasmin which activate metalloprotease that destroy basement membrane
7-produce heat shock protein 70 involved in survive
8- associated with biofilm
Naegleria fowleri
Pathogenesis and viruleince factors
The portal of entry into the CNS is the olfactory neuroepithelium. It is believed that the sustentacular cells lining the olfactory neuroepithelium phagocytose the amoebae that enter the nasal passa

1. 1 st Lab. Genetic Materials Assist. Prof. Dr. Mohammed Abbas Jasim Microbiology Dept./ College of Medicine University of Anbar

2. Introduction DNA isolation is an essential technique in molecular biology. Isolation of high- molecular weight DNA has become very important with the increasing demand for DNA fingerprinting, restriction fragment length polymorphism (RFLP), construction of genomic or sequencing libraries and PCR analysis in research laboratories and industry. Also, DNA isolation is the first step in the study of specific DNA sequences within a complex DNA population, and in the analysis of genome structure and gene expression. The quantity, quality and integrity of DNA will directly affect these results. DNA constitutes a small percentage of the cell material and is usually localized in a defined part of the cell.

3. In prokaryotic cells, DNA is localized in the nucleoid that is not separated from the rest of the cell sap by a membrane. In eukaryotic cells, the bulk of DNA is localized in the nucleus, an organelle that is separated from the cytoplasm by a membrane. The nucleus contains about 90% of the total cellular DNA, the remaining DNA is in other organelles like mitochondria or chloroplasts. In viruses and bacteriophages, the DNA is encapsulated by a protein coat, and constitutes between 30 and 50 percent of the total mass of the virion. In prokaryotic and eukaryotic cells, DNA constitutes only about 1% of the total mass of the cell. The approximate composition of rapidly dividing E.coli cells and human cells (HeLa) is presented in Table 1.

4. •

5. The purpose of DNA isolation is to separate DNA from all the components of the cell listed in Table 1 resulting in a homogeneous DNA preparation that represents the entire genetic information contained within a cell. There is no difficulty in separating DNA from small molecules since the molecular weight of DNA is very large. Consequently, the main cellular components that have to be removed during DNA purification are protein and RNA.

6. General requirements of effective DNA isolation The method should yield DNA without major contaminants, protein and RNA. The method should be efficient; most of the cellular DNA should be isolated and purified. It also should be nonselective; all species of DNA in the cells should be purified with equal efficiency. The method should not physically or chemically alter DNA molecules. The DNA obtained should be of high molecular weight and with few single-stranded breaks. The method should be relatively fast and simple enough that it will not take a long time or much effort to prepare DNA. This is important since preparation of DNA is just the beginning of an experiment and not an end in itself.

7. There are several methods for isolation of DNA that, in general, fulfill most of the requirements listed above. All methods involve four essential steps: Cell breakage. Removal of protein and RNA. Concentration of DNA. Determination of the purity and quantity of DNA. Two of the most common obstacles in obtaining a high yield of high molecular weight DNA are hydrodynamic shearing and DNA degradation by nonspecific DNases. To avoid these problems, some general precautions should be taken. All solutions should contain DNase inhibitors and all glassware, plastic pipettes tips, centrifuge tubes, and buffers should be sterilized. The method of cell breakage used should avoid strong forces that shear the DNA. DNA, in solution, should always be pipetted slowly with wide- bore pipettes (about 3 to 4 mm orifice diameter). The tip of the pipette should always be immersed in the liquid when pipetting DNA. The DNA solution should never be allowed to run down the side of a tube nor should it be vigorously shaken or vortexed. The use of molecular biology grade or ultrapure chemical reagents is strongly recommended.

8. Each DNA purification task should begin with careful planning of the amount of DNA needed, the purity required and an estimation of the size of DNA molecules needed. Preparation of a genomic library requires 100 to 300 ug of large molecular weight DNA (more than 100000 bp long), essentially devoid of protein contamination. Southern blot analysis of a single copy gene requires 5 to 10 Ilg of eukaryotic, genomic DNA per single gel lane.

9. The purity and the size of this DNA is not as critical as it is for genomic. library preparation. DNA of 50000 to 100 000 bp is sufficient for most applications with purity achieved by standard DNA isolation methods. A single PCR reaction requires only a very small amount of DNA (50 to 500 ng) and can tolerate a considerable amount of contaminating proteins. Indeed, it is possible to achieve PCR amplification of DNA from a single lysed cell. However, care must be taken to remove excess RNA from the DNA preparation, because a large amount of RNA can severely inhibit the PCR reaction

10. ISOLATION AND SEPARATION OF NUCLEIC ACIDS Isolation of DNA The use of DNA for analysis or manipulation usually requires that it is isolated and purified to a certain extent. DNA is recovered from cells by the gentlest possible method of cell rupture to prevent the DNA from fragmenting by mechanical shearing. This is usually in the presence of EDTA which chelate the Mg 2+ ions needed for enzymes that degrade DNA termed DNase. Ideally, cell walls, if present, should be digested enzymatically (e.g. lysozyme treatment of bacteria) and the cell membrane should be solubilised using detergent. If physical disruption is necessary, it should be kept to a minimum and should involve cutting or squashing of cells, rather than the use of shear forces. Cell disruption (and most subsequent steps) should be performed at 4 C, using glassware and solutions which have been autoclaved to destroy DNase activity.

11. After release of nucleic acids from the cells, RNA can be removed by treatment with ribonuclease (RNase) which has been heat treated to inactivate any DNase contaminants; RNase is relatively stable to heat as a result of its disulfide bonds, which ensure rapid renaturation of the molecule on cooling. The other major contaminant, protein, is removed by shaking the solution gently with water- saturated phenol or with a phenol–chloroform mixture, either of which will denature proteins but not nucleic acids. Centrifugation of the emulsion formed by this mixing produces a lower, organic phase, separated from the upper, aqueous phase by an interface of denatured protein. The aqueous solution is recovered and deproteinised repeatedly, until no more material is seen at the interface. Finally, the deproteinised DNA preparation is mixed with two volumes of absolute ethanol and the DNA allowed to precipitate out of solution in a freezer. After centrifugation, the DNA pellet is redissolved in a buffer containing EDTA to inactivate any Dnases present. This solution can be stored at 4 C for at least 1 month. DNA solutions can be stored frozen, although repeated freezing and thawing tend to damage long DNA molecules by shearing. •

12. Flow diagram of the main steps involved in the extraction of DNA.

13. It is possible to check the integrity of the DNA by agarose gel electrophoresis and determine the concentration of the DNA by using the fact that 1 absorbance unit equates to 50 mg ml1 of DNA and so Contaminants may also be identified by scanning UV spectrophotometry from 200 to 300 nm. A ratio of 260 nm:280 nm of approximately 1.8 indicates that the sample is free of protein contamination, which absorbs strongly at 280 nm.

14. Isolation of RNA The methods used for RNA isolation are very similar to those described above for DNA; however, RNA molecules are relatively short and therefore less easily damaged by shearing, so cell disruption can be more vigorous. RNA is, however, very vulnerable to digestion by Rnases which are present endogenously in various concentrations in certain cell types and exogenously on fingers. Gloves should therefore be worn and a strong detergent should be included in the isolation medium to denature immediately any RNases. Subsequent deproteinisation should be particularly rigorous, since RNA is often tightly associated with proteins. DNase treatment can be used to remove DNA and RNA can be precipitated by ethanol. One reagent in which is commonly used in RNA extraction is guanadinium thiocyanate, which is both a strong inhibitor of RNase and a protein denaturant.

15. It is possible to check the integrity of an RNA extract by analyzing it by agarose gel electrophoresis. The most abundant RNA species are rRNA molecules, 23S and 16S for prokaryotes and 18S and 28S for eukaryotes. These appear as discrete bands on the agarose gel and indicate that the other RNA components are likely to be intact. This is usually carried out under denaturing conditions to prevent secondary structure formation in the RNA. The concentration of the RNA may be estimated by using UV spectrophotometry. At 260 nm, 1 absorbance unit equates to 40 mg mlof RNA and therefore

16. Contaminants may also be identified in the same way as for DNA by scanning UV spectrophotometry; however, in the case of RNA a 260 nm:280 nm ratio of approximately 2 would be expected for a sample free of contamination. In many cases, it is desirable to isolate eukaryotic mRNA, which constitutes only 2–5% of cellular RNA, from a mixture of total RNA molecules. This may be carried out by affinity chromatography on oligo (dT)-cellulose columns. At high salt concentrations, the mRNA containing poly(A) tails binds to the complementary oligo(dT) molecules of the affinity column and so mRNA will be retained; all other RNA molecules can be washed through the column with further high-salt solution. Finally, the bound mRNA can be eluted using a low concentration of salt. Nucleic acid species may also be subfractionated by more physical means such as electrophoretic or chromatographic separations based on differences in nucleic acid fragment sizes or physicochemical characteristics.

17. Electrophoresis of nucleic acids Electrophoresis in agarose or polyacrylamide gels is the most usual way to separate DNA molecules according to size . The technique can be used analytically or preparatively and can be qualitative or quantitative. Large fragments of DNA such as chromosomes may also be separated by a modification of electrophoresis termed pulsed field gel electrophoresis (PFGE). The easiest and most widely applicable method is electrophoresis in horizontal agarose gels, followed by staining with ethidium bromide. This dye binds to DNA by insertion between stacked base pairs (intercalation) and it exhibits a strong orange/red fluorescence when illuminated with ultraviolet light. Very often electrophoresis is used to check the purity and intactness of a DNA preparation or to assess the extent of a enzymatic reaction during, for example, the steps involved in the cloning of DNA.

18. Agarose gel electrophoresis of DNA Agarose gels can be used to separate molecules larger than about 100 bp. For higher resolution or for the effective separation of shorter DNA molecules, polyacrylamide gels are the preferred method

19. When electrophoresis is used preparatively, the piece of gel containing the desired DNA fragment is physically removed with a scalpel. The DNA may be recovered from the gel fragment in various ways. This may include crushing with a glass rod in a small volume of buffer, using agarase to digest the agarose thus leaving the DNA, or by the process of electroelution. In the latter method, the piece of gel is sealed in a length of dialysis tubing containing buffer and is then placed between two electrodes in a tank containing more buffer. Passage of an electric current between the electrodes causes DNA to migrate out of the gel piece, but it remains trapped within the dialysis tubing and can therefore be recovered easily

20. More commonly, commercial spin columns can be used which contain an isolating matrix used in conjunction with a bench-top microcentrifuge. The use of such standardised ‘kits’ in molecular biology is now common place. An alternative to conventional analysis of nucleic acids by electrophoresis is through the use of microfluidic systems. These are carefully manufactured chip-based units where microlitre volumes may be used and with the aid of computer analysis provide much of the data necessary for analysis. Their advantage lies in the fact that the sample volume is very small, allowing much of an extract to be used for further analysis

21. End of Lecture