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.  

What is Bacteriophage Artificial Chromosomes (BACs) ?

What is Bacteriophage Artificial Chromosomes (BACs) ?

These plasmids are circular DNA molecules carrying conventional antibiotic resistance marker, origin of replication derived from the F factor of E.coli, an ATP driven helicase (repE) to facilitate DNA replication and three loci (parA, parB and parC) for proper partitioning of the plasmid to daughter cells. BAC vectors have no packing constraints and there is no fixed limit to the size of genomic DNA that they accept. Usually the size of DNA is approximately 120-kilo base pairs.  

what is Shuttle vector in gene cloning ?

What is Shuttle vector in gene cloning ?

Cloning of foreign DNA is usually carried out primarily in E.coli since the organism is most thoroughly studied. But subsequent work often requires the foreign segment to be delivered to different host cells like eukaryotes. A number of vectors are devised to satisfy this requirement. These vectors are termed as shuttle vectors. These vectors have origins of replication of various hosts. The also contain fragments of eukaryotic viruses to facilitate entry into the cell or expression or integration in the cell itself. Thus shuttle vectors allow DNA to be transferred between two different species where it can be propagated by utilizing both the origins of replication. Usually the origins of replication are derived from bacterial and eukaryotic systems. Shuttle vectors also carry antibiotic resistance genes, which are functional in eukaryotes e.g. Neomycin (G418), Hygromycin, Methotrexate etc. All the DNA manipulation and characterization are done in prokaryotic system and then the manipulated DNA is introduced into the eukaryotic systems for protein expression and functional analysis. Eukaryotic host systems are better for expression of protein for few reasons:

1. Proper folding of the protein to attain functional activity .

2. Posttranslational modification of proteins for which prokaryotes does not possess any machinery. The most conventional and convenient model system for expression of eukaryotic proteins is yeast, Pichia pastoris, which is both genetically and physiologically well characterized. 

Yeast Artificial Chromosome (YACs)

Yeast Artificial Chromosome (YACs)

These are linear DNA molecules similar to yeast chromosome. Recombinant YACs are made by ligating large fragments of genomic DNA and then the resultant plasmid is introduced into yeast by transformation. The vector carries selection marker, DNA sequences called as telomere, so that the product can be stabilized inside the yeast cell, an origin of replication called autonomous replication origin, ARS. Large size of DNA can be inserted into YAC vectors, usually between 250kilobases to 400kilobasepairs. Large size of mammalian genomic libraries is also made with approximately 1 megabasepairs of foreign inserts. Insertion of foreign DNA into the cloning site inactivates a mutant expressed in vector DNA and formation of red rather than white colonies by yeast strain is observed. Thus transformants are identified as red colonies, which grow in yeast that is mutant for TRP1 and URA3, which ensure that the cell has received an artificial chromosome and with both the telomeres since it is complimented for both the mutations. And the colony also contains foreign DNA because it is red in color.

Brief note about plant vector

What is plant vector ?

Plant Vectors

A vector is a circular DNA molecule capable of independent existence and replication within a host cell. In case of plants, Ti and Ri are the two most commonly used plasmids which are used as vectors. Plant cells as such do not possess any endogenous plasmids. But two plasmids called pTi and pRi, are present naturally in the bacteria, Agrobacterium tumefaciens and Agrobacterium rhizogenes, respectively. These plasmids provide a naturally occurring transformation system. A part of the plasmid DNA, called as T-DNA, is transferred into the genomes of most dicot and some monocot plants. pTi stands for tumor inducing plasmid and pRi stands for root inducing plasmid. The infection of Agrobacterium tumefaciens is mediated by transfer of a segment of pTi called as T-DNA into the plant cell. Various bacterial chromosomal genes, such as chvB, exo genes, cell genes, are concerned with the biosynthesis of cell attachment polysaccharides due to which the bacterial cells adhere firmly to the plant cells. While two chromosomal genes are expressed constitutively in bacterial cells that is expressed at all the times inside a cell, which are responsible for virulence associated aspects, Agrobacterium tumefaciens Ti plasmid produces tumor like growth from which roots / shots may sometimes be produced. The infected cells are able to grow in culture on a medium devoid of any growth regulator while uninfected normal plant cells need exogenous auxin or cytokinin. These plasmids also carry genes for IAA (Indole Acetic Acid - auxin) and cytokinin production which is the reason for indefinite growth on a growth regulator free culture medium. When pTi is introduced into Rhizobium trifolii, it gains the ability to produce galls and to utilize opines. The crown gall root cells also synthesize unique nitrogenous compounds called opines, which are not produced by normal plant cells, which are not infected, nor are they utilized. The infected cells use opines as their carbon and nitrogen source. The type of opine produced depends on the bacterial strain. Agrobacterium tunefaciens strain produces either octopine or nopaline which the Agrobacterium rhizogenes produce either agropine or mannopine.

Brief note about Ti-plasmid

Brief note about  Ti plasmid

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Ti plasmid is a large mega plasmid conjugative plasmid of ~200kb. pTi is lost when Agrobacterium is grown above 28oC, such cured bacteria do not induce crown galls that is, they become a virulent. pTi and pRi, although do not share sequence homology but are unique in following respects:- a) They contain some genes, which are located within their T-DNA which has regulatory sequences recognized by plant cells, while their remaining genes have prokaryotic regulatory sequences. As a result, the former are expressed only in plant cells but not in the Agrobacterium, while the latter are only expressed in the bacterium. b) These plasmids naturally transfer a part of their DNA, called as T-DNA, into host plant cells. The T-DNA usually contains following important functional regions.
1. T-DNA contains oncogenes and opine synthesis genes and is transferred into host plant.
2. Vir region which regulates the transfer of T-DNA
3. Opine catabolism genes for utilization of opines.
4. Origin of replication for propagation in Agrobacterium. The T-DNA contains a 24bp direct repeat border sequence and contains the genes necessary for tumor / possess gene for auxin and cytokinin biosynthesis. All the genes present in TDNA have eukaryotic regulatory sequences. As a result, these genes are expressed only in plant cells but never express in Agrobacterium. The vir region mediates the transfer of TDNA into plant genomes and hence is essential for virulence. The genes of vir region are not transferred but induce the transfer of T-DNA. Also, the genes present in T-DNA are not responsible for its transfer, but the 24 bp direct repeat at both the left and right ends of TDNA is essential for the transfer. The exact mechanism of transfer of T-DNA is not known clearly known but is brought by the vir region. The phenols produced by wounded plant tissue initiates the transfer process. The T-DNA is transferred into the plant cells as single stranded DNA, which increases the efficiency of its transformation. But, as soon as it enters into the plant cell, it is immediately converted into a double stranded form. This form integrates at random sites in the host plant genome by a phenomenon called illegitimate recombination, which are due to sequence of homology in short segments of the host DNA. This integration is usually in low copy numbers. Few vectors are derived from pTi (wild type) due to some problems posed by wild type plasmid eg. The presence of oncogenes causes a disorganized growth, their large size and lack of cloning sites within the T-DNA, which are needed for the insertion of DNA segments that has to be cloned. 

What is Disarming ? Or removal of oncogenes from T-DNA into plant cell. & What is the Common Method of gene trnasfar

What is Disarming ? Or removal of oncogenes from T-DNA into plant cell.

What is the Common Method of gene transfar?

Disarming is the removal of oncogenes from T-DNA and it has resolved many problems. This disarmed plasmid can still transfer its T-DNA into plant cells. But the cells containing this modified T-DNA will be non tumorous, produce opines and generate plantlets. Only the border sequences are necessary for the transfer of any DNA insert placed between them. Thus pTi and pRi which are disarmed are more in use for gene transfer experiments. But in some plants, the efficiency of transformation by disarmed pTi is much lower than the wild type pTi.  Introduction of DNA into plant cells without the involvement of a biological agent like Agrobacterium leading to a stable transformation is known as Direct Gene Transfer. The spontaneous uptake of DNA is quite low.

The various methods those are utilized for direct gene transfer are

1. Chemical methods like PEG, Calcium phosphate
2. Electroporation
3. Particle gun delivery
4. Lipofection
5. Microinjection and Macroinjection

What is Ethidium bromide?

What is Ethidium bromide?

Answer- Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels. It is added to the DNA sample before loading to enable visualization of the fragments within the gel or can be added in the electrophoresis buffer. The binding of ethidium bromide to DNA alters its mass and rigidity, and therefore its mobility. 

What is phage virus ?

What is phage ?

PHAGE can also replicate via the Lysogenic cycle. The phage genome is integrated into the host chromosome and is inherited into the chromosomes of all daughter bacteria. This "prophage" can be induced to enter the lytic cycle and kill its host by a variety of stresses like UV light

What is the Bacteriophage?

What is Bacteriophage ?

Bacteriophages replicate via the lytic phase cycle and the phage genome is injected into the cell, phage genes are expressed and phage proteins and DNA are made, progeny phage are packaged, and the cell is lysed. Two genetically different phage that infect the same host cell may recombine during the lytic cycle

What is Recombinant DNA Technology in brief

What is a Recombinant DNA ?

DNA molecules constructed outside the living cells that is in vitro by joining natural or synthetic DNA segments that can replicate in a living cell.

What is the Goals of Recombinant DNA Technology?

a) To isolate and characterize a gene
b) To make desired alterations in one or more isolated genes
c) To return altered genes to living cells

What is Basic Tools of Recombinant DNA Technology?

Nucleic Acid Enzymes DNA and RNA polymerases, reverse transcriptase, DNA ligases, Restriction endonucleases and many more.

How to isolate a plasmid from a bacteria ?

How to isolate a plasmid from a bacteria ?

Plasmids are easily isolated from bacterial cells Plasmid isolation takes advantage of the unique structural properties of plasmids. Plasmids are small,super coiled circular pieces of Dana.Unlike the much larger bacterial chromosomes(which is also circular),plasmids are quite resistant to permanent den. Today,most laboratories use commercial kits for plea mid isolations,because the kits are convenient and relatively inexpensive.the kits give good yields of highway a little,while avoiding the need for organic denaturant ants.A variety of less expensive,but somewhat more time-consuming,procedure have been described for investigators who want to make their own reagents.These procedures generally give good yields of death at is slightly less pure in a purified with the kits.Whatever the isolation procedure,the general principles of plasmid isolation are the same.The figure and paragraphs below summarise regs general principles used for plasmid isolation.

1. Lysis and denaturation-

Strong denaturating conditions to weaken the tough bacterial drop wall.The most common procedures use a combination of strong base and a detergent.The detergents help to solubility lipids in the,allowing the denaturants to enter the cell. Proteins,because of their fragile structures,are irreversibly denatured.The treatment also breaks the hydrogen bonds holding together the chromosomal and plasmid DNA.

 2. Neutralization-

Neutralization allows complementary DNA strand store anneal and causes proteins to precipitate.Plasmids renature because they have super coiled structures that have held the two strands of the helix together denaturation.chromosomal DNA a is not able to renature,however,because it's longer strands have become mixed with denatured proteins. Samples must be mixed gently at this step to prevent fragmentation of the long, chromosomal DNA into pieces that might be able to reanneal and co-purify with the plasmids.  

3. centrifugal- 

on plasmid Dana is separated from large aggregates of precipitated protea chromosomal in a by centrifugal ion. 

4. Additional purification -

Plasmids are further purified by organic extraction or adsorption in resin.

What is plasmid DNA?

What is plasmid DNA?

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Plasmid is a double standed ,self replicating,extra chromosomal DNA Plasmids are cloning vectors that are widely used in molecularbiology andthey play an important roles in the laboratory.Plasmids are small, circular pieces of DNA  that replicate independently of the host chromosome.The first plasmids used in the lab were derivatives of naturally I occurring plasmids found in vacate.Since their discovery,molecularbiologists have added many features to plasmids to suit a variety of applications.In this lab,each team will I so late three plasmids from bacterial strains.

Plasmid DNA are red circular one

a short notes on Insulin of human body

                                       Human Insulin 

Human insulin is a globular protein with a molecular weight of about 5,800 kd, consisting of 51 aminoacid residues organised in two polypeptide chains (A and B), linked by two disulphide bonds. Chain A consists of 21 residues with an extra disulphide bond between A6 and A11; chain B consists of 30 aminoacids. Complete synthesis of the human insulin molecule was achieved in 196610. Insulin exists as a monomer only at low concentrations while it shows propensity to aggregate into stable dimers at higher concentrations, in aqueous solution at pH 2-8 and into hexamers in the presence of zinc ions. The hexamer, in which chain A constitutes much of the polar surface, is almost spherical in structure, with a diameter of 5 nm and a height of 3.5 nm. Polymerisation of the hormone has major pharmacological implications.

What Is Cancer?

 What Is Cancer?

=> Cancer results from a series of molecular events that fundamentally alter the normal properties of cells. In cancer cells the normal control systems that prevent cell overgrowth and the invasion of other tissues are disabled. These altered cells divide and grow in the presence of signals that normally inhibit cell growth; therefore, they no longer require special signals to induce cell growth and division. As these cells grow they develop new characteristics, including changes in cell structure, decreased cell adhesion, and production of new enzymes. These heritable changes allow the cell and its progeny to divide and grow, even in the presence of normal cells that typically inhibit the growth of nearby cells. Such changes allow the cancer cells to spread and invade other tissues. The abnormalities in cancer cells usually result from mutations in protein-encoding genes that regulate cell division. Over time more genes become mutated. This is often because the genes that make the proteins that normally repair DNA damage are themselves not functioning normally because they are also mutated. Consequently, mutations begin to increase in the cell, causing further abnormalities in that cell and the daughter cells. Some of these mutated cells die, but other alterations may give the abnormal cell a selective advantage that allows it to multiply much more rapidly than the normal cells. This enhanced growth describes most cancer cells, which have gained functions repressed in the normal, healthy cells. As long as these cells remain in their original location, they are considered benign; if they become invasive, they are considered malignant. Cancer cells in malignant tumors can often metastasize, sending cancer cells to distant sites in the body where new tumors may form.

What is a species?

What is a species?

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 The most common definition is based on the biological species concept. By this definition, individuals belong to the same species if they reproduce by mating with each other and are reproductively isolated from other such groups.

How to prepare ACETIC ACID- SODIUM ACETATE BUFFER?

(This is practical experiment)

How to prepare ACETIC ACID- SODIUM ACETATE BUFFER?

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 REAGENTS REQUIRED:  

Acetic Acid 0.2M: 1.5 ml of glacial acetic acid is made upto 100ml with     distilled water. 

Sodium Acetate Solution: 0.64 gm of sodium acetate or 2.72gm of sodium acetate trihydrate is dissolved in 100ml Distilled water. 

PROCEDURE: 

Pipette out exactly 36.2ml of sodium acetate solution into 100ml of standard flask and add 14.8ml of glacial acetic acid, make the volume 100ml using distilled water using distilled water. This gives 0.2 M of acetic acid and sodium acetate buffer. The pH is measured with pH meter.  

 pH meter is first standararised with pH buffer. Wash electrode with distilled water and introduced into 0.2M acetic acid-sodium acetate buffer prepared, the pH of solution is 4.6. 

 RESULT:  

36.2ml Sodium acetate and 14.8 ml glacial acetic acid were mixed and buffer was prepared. pH was measured initial reading observed was 4 which made upto 4.6 with 5N NaOH.   

How to prepare BARBITONE BUFFER?

(This is practical experiment)

How to prepare BARBITONE BUFFER?

  REAGENTS REQUIRED:

• Diethyl barbituric acid. 

• Sodium diethyl barbititrate 

PROCEDURE:   

Dissolve 2.85gm of diethyl barbituric acid and 14.2gm of sodium diethyl barbititrate in distilled water and upto 1 liter. This gives the barbitone buffer.  The pH meter is first standararised with pH buffer. Wash electrode with distilled water and introduced into barbitone buffer prepared, the pH of solution is 6.8.