HIV is a complex animal virus

HIV is a complex animal virus

AIDS

The animal virus HIV infects certain key cells of the immune system, destroying the ability of the body to defend itself from  cancer and disease.
The HIV infection cycle is typically a lytic cycle, in which the HIV RNA first directs the production of a corresponding DNA, and this DNA then directs the production of progeny virus particles. The Future of HIV Treatment.
Combination therapies and chemokines offer promising avenues of AIDS therapy.

What is Disease Viruses ? Who are they?

What is Disease Viruses ? Who are they?

Humans have known and feared diseases caused by viruses for thousands of years. Among the diseases that viruses cause are influenza, smallpox, infectious hepatitis, yellow fever, polio, rabies, and AIDS, as well as many other diseases not as well known. In addition, viruses have been implicated in some cancers and leukemias. For many autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis, and for diabetes, specific viruses have been found associated with certain cases.

In view of their effects, it is easy to see why the late Sir Peter Medawar, Nobel laureate in Physiology or Medicine, wrote, “A virus is a piece of bad news wrapped in protein.”
Viruses not only cause many human diseases, but also cause major losses in agriculture, forestry, and in the productivity of natural ecosystems.

What is Viroids?

What is Viroids?

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Viroids are tiny, naked molecules of RNA, only a few hundred nucleotides long, that are important infectious disease agents in plants. A recent viroid outbreak killed over ten million coconut palms in the Philippines. It is not clear how viroids cause disease. One clue is that viroid nucleotide sequences resemble the sequences of introns within ribosomal RNA genes. These sequences are capable of catalyzing excision from DNA—perhaps the viroids are catalyzing the destruction of chromosomal integrity.

Taq DNA polymerase

Taq DNA polymerase:-


  Taq DNA polymerase is a DNA dependent DNA polymerase, first isolated from the hot spring bacterium, Thermus aquatics in 1976 and in 1989. Due to its wide use in molecular biology (primarily PCR), it is termed as ‘Molecule of the Year’. This thermophilic DNA polymerase encodes an 832-amino acid, 94 kDa protein, which consists of two domains.

1. -NH2 domain:  Similar to 5’-3’ exonuclease domain of members of polymerase I family of DNA polymerase

2. -C terminal domain contains a catalytically inactive 3’-5’ exonuclease and a polymerase sub domain, similar to klenow of DNA polymerase I.

The thermal stability of Taq DNA polymerase is attributed to its hydrophobic core and stable electrostatic interactions and high density of proline residues on the surface of the enzyme.

The optimal activity is at 75-800C temperature and at 600C, the activity is reduced by a factor of 2 and at 370C, its activity is reduced to only 10%.

To initiate DNA synthesis, like other DNA polymerases, it also requires a primer that is annealed to the template strand and caries an extensible 3’-OH group.

Taq DNA polymerase requires Mg2+ for its optimal activity. Phosphate buffers inhibit Taq DNA polymerase and therefore should be avoided. The reaction is usually carried out in the presence of Tris buffer at pH 8.3. Because of the lack of a proofreading function, the rate of misincorporation of dNTPs is high in PCR reactions which are catalyzed by Taq polymerase (or any other DNA polymerase that does not have editing domains).
Several mutant forms of native polymerases, are also available like Pfu And vent. Both of them have proofreading activities contributed by 3’-5’ exonuclease.

 Pfu polymerase is therefore known to generate lowest errors while vent is probably intermediate between Taq and Pfu.

 Pfu polymerase is isolated from Pyrococus furiosus Vent is isolated from Thermococcus litoralis (also known as Tli polymerase) 

T7 DNA Polymerase

T7 DNA Polymerase



 The T7 DNA polymerase from T7 bacteriophage has 3’-5’ exonuclease and DNA polymerase activity but lacks 5’-3’ exonuclease domain, which is similar to T4 DNA polymerase. The processivity of this enzyme is quite good that is, the average length of DNA synthesized before the enzyme dissociates from the template, is considerably greater than for other enzymes. Thus, the average length of DNA synthesized by a single molecule of bacteriophage T7 polymerase is much greater than that of DNAs synthesized by other DNA polymerases. The binding and polymerization domain is occupied by the carboxy terminus while the potent 3’-5’exonuclease activity resides on the amino terminus.

The exonuclease activity is completed inactivated by incubating the enzyme with a reducing agent, molecular oxygen, and low concentrations of ferrous ions, for several days. Over 99% of the exonuclease activity is abolished without affecting the polymerization activity by these agents, which cause mutations and site specific modifications. The resulting chemically modified enzyme is marketed under the trade name Sequenase is ideal for determining the sequence of long tracts of DNA by the dideoxy mediated chain termination method. 

Insertion Vectors

Insertion Vectors

Vectors that have a single target site for insertion of foreign DNA are known as insertion vectors. 20% DNA that is not required for lytic growth is removed and therefore insertion of foreign DNA resumes the size back to something like its full length and can be packaged in vitro. Maximum size of DNA that can be accommodated varies from 9- 11 kb. For DNA of larger sizes, high capacity vectors are designed like:-

Vector                size.                     OrI                            Host
Cosmid.            30-45kb.           Col E1                           E.coli
BAC.                 120-300kb.          Replicon forgein      E.coli  
YAC                   250-400kb           ARS                            Yeast

PCR Mediated Gene Cloning

PCR Mediated Gene Cloning

 There are three strategies for cloning PCR products-

 1) T/A cloning is the easiest cloning method. T/A cloning takes advantage of the terminal transferase activity of Taq polymerase and other non-proofreading DNA polymerases which adds a single 3'-A overhang to each end of the PCR product. The resulting PCR product is then ligated into a linear vector with a 3´ terminal 'T' or 'U' at both ends.

 2) Directional cloning. A restriction enzyme target site is introduced into each of the PCR primers. The resulting PCR product and cloning vector are digested with the restriction enzymes to generate complementary ends at the PCR product and the vector which are then ligated.

  3) Blunt-end cloning-Blunt-end PCR product generated by proof-reading polymerase such as the Pfu DNA Polymerase can also be cloned into a blunt-end vector.

The cloning of PCR-amplified fragments into a linear vector is typically a rapid and efficient process. However, not all PCR fragments will clone with the same efficiency into the same vector. These differences may be due to fragment size, insert toxicity, and the complexity of the insert. The size of the fragment being cloned is a primary contributor to the overall cloning efficiency. Large fragments of DNA (≥ 5 kb) are amenable to cloning in high-copy number vectors, yet at a much lower efficiency. Optimization of molar concentration ratios of the vector to insert is critical to ensure efficient cloning. Successful cloning ratios may range from 1:1 to 1:10. For example, if the vector is 3 kb and the insert is 1 kb, one-third the amount of insert needs to be added to attain a 1:1 molar ratio.