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 the Conditions and requirements that influence Enzyme Activity for the use of Restriction Endonuclease at the time of treatment with plasmid ?

What is the Conditions and requirements that influence Enzyme Activity for the use of Restriction Endonuclease at the time of treatment with plasmid ?

One unit activity :-

it is defined as amount of enzyme required to digest 1µg of reference DNA in 60 minutes at 370C. Usually reference DNA is λ phage DNA.

Buffer:- 

The most critical determinant of the enzyme activity is the ionic concentration (NaCl content) of the buffer. There are commercially 3 buffer systems available for the activity, low salt buffer (20mM), medium (100mM) or high (250mM) NaCl buffers. The buffer is usually 10mM Tris-HCl, pH 8.0, supplemented with Magnesium salt (often 50mM MgCl2), a reducing agent (usually 1mM DTT), and some enzymes require BSA (100µg/ml) and salt (NaCl). The reaction does not require ATP.

Quality of DNA :-

Preparation of DNA that has to be cleaved should be free of contaminants such as phenol, CHCl3, alcohol, EDTA, detergents, excessive salts, protein, RNA, all of which can pose a hindrance to the enzyme’s activity.

Stopping a Reaction :

To terminate Restriction enzymes activity, heat inactivation is a way but for the enzymes which it does not work, phenol / CHCl3 extraction is another means of inactivation of Restriction enzymes.

Incubation Temperature and Time  : - 

Recommended temperature for most of the Restriction enzymes is 370C except for the ones which are isolated from thermophilic bacteria which require higher temperature ranging from 50-650C. While some restriction enzymes have short ½ lives at 370C and for those lower temperatures are required. Incubation time as per the unit definition is 1 hour but it can be manipulated. Example, it may be shortened if more units of Restriction enzyme is added or longer times are used to allow a reaction to proceed to completion with fewer enzymes units.

What is electrophoresis buffer ?

What is Electrophoresis buffer?

Several different buffers have been recommended for electrophoresis of DNA. The most commonly used for duplex DNA are TAE (Tris-acetateEDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate at somewhat different rates in these two buffers due to differences in ionic strength. Buffers not only establish a pH, but provide ions to support conductivity. If water is used instead of buffer there will be no migration of DNA in the gel and conversely, if concentrated buffer is used like a 10X solution instead of 1X, heat will be generated in the gel which is enough to melt it. 

What is Isochizomers

What is Isochizomers?

Isochizomers are different Restriction endonucleases having same recognition site. In some cases, they cut identically within their recognition site, but sometime they do not. They have different optimum reaction conditions, stabilities and cost that give us an option of what to purchase. Some Restriction endonucleases recognizes only one sequence but never other, called as Ambiguous Recognition Sequence. Eg. BamH I recognize GGATCC, while Hinf I recognizes a 5bp sequence, with an eligibility of sequence starting with GA and ending with TC and having any base in between GANTC. Some REs recognition site has a site for cleavage by other Restriction Endonuclease. e.g: BamH I site GGATCC have site recognized and cleaved by Sau3A I GATC. Thus all BamH I sites can consequently be cut by Sau3A I.

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.