To Cleave Plasmid and Lambda Dna Using Restriction Enzyme

Lab Report – 102 and 105

Objective:

102: To cleave Plasmid and Lambda DNA using restriction enzyme

105: To map the restriction sites on Plasmid DNA

Background information:

Restriction enzymes: They are type of enzymes originally isolated form bacteria and archaea.  Over 2,100 restriction enzymes have been discovered and catalogued till date. Restriction enzymes are endonucleases that cleave both strands of DNA at near specific recognition sequences known as restriction sites. They scan a DNA molecule, looking for a particular sequence, usually of four to six nucleotides once they find this recognition sequence; they stop and cut the phosphodiester bond in the backbone of DNA. This produces a 5’ phosphate and 3’ hydroxyl group. Many enzymes require cofactors like Mg2+ to function.  The resulting DNA fragments are separated using electrophoresis. They result in covalent bond (within a single strand) and a hydrogen bond (between strands) when bonds are broken. They are also called as molecular scissors.

There are four types of restriction enzymes:

Type I: It cleaves sites remote from recognition sites and it requires ATP for enzymatic activity.

Type II: It cleaves within a short specific recognition palindromic sequence (4-8bp), and it requires Mg2+ to function.  It is most widely used restriction enzyme for recombinant DNA technology and gene mapping type due its ability to cut at highly specific sites. Based on their recognition site they restriction enzyme can either produce a sticky end (EcoRI, BamHI ) or a blunt end (SmaI, PbuII).

Type III: It cleaves at a short distance from the recognition site and it requires ATP for enzymatic activity.

Type IV: It recognizes and cleaves modified, typically methylated DNA.

Each enzyme is named after the bacterium from which it was isolated, using a naming system based on bacterial genus, species and strain. For example: EcoRI – E (Genus Escherichia), co (species coli), R (strain RY13), I (first identified).

Plasmid DNA: Plasmids are circular fragments of double-stranded DNA that can replicate independently of chromosomal DNA. They are 1 to over 500 Kbp. There are 4 types of plasmid conformations; Supercoiled, nicked and relaxed

Lambda DNA: Lambda DNA is phage of the lambda virus. They are 48,000bp. It is usually used as standard marker for DNA. It is usually linear in conformation.

Gel Electrophoresis: We use gel electrophoresis to learn deeply about DNA structure and function. We apply an electrical charge, pushing the negatively charged molecules towards the positive side. The smaller the molecule, the less resistance it will face when hitting the pores of the gel, and the farther it will travel. Nucleic acids, both DNA and RNA, can be separated on the basis of size and charge by means of electrophoresis to identify structural forms of plasmid DNA or Lambda DNA, as well as determine the size of DNA fragments of genes. Furthermore, the size of unknown DNA fragments can be determined be constructing a standard curve using the migration distances and sizes of a known DNA marker. We used agarose (originally derived from seaweed) in the gel as Agarose gels have a very large “pore” size and are used primarily to separate very large molecules such as DNA.

Results:

1. Plasmid DNA uncut
2. Plasmid DNA cut with BgII
3. Plasmid DNA cut with EcoRI
4. Lambda DNA uncut
5. Lambda DNA cut with EcoRI
6. Plasmid DNA cut with enzyme 1
7. Plasmid DNA cut with enzyme 2
8. Plasmid DNA cut with enzyme 1 and 2

M. Marker

Interpretation and Discussion of Results:

1. Marker

[pic 1]

In the above graph Y represents log DNA fragments size and x represents the distance of migration by the DNA. Hence we are able to calculate the size of other DNA fragments by the function we get using the above graph.

1. Analysis of A, B & C

A: Plasmid DNA Uncut

It can be seen that there are two bands in lane A mean there are two forms of plasmid, one is super coil DNA (3311 bp) and one is linear DNA (5248 bp).

B: Plasmid DNA Cut with BglI

It can clearly seen that there is only one band in lane B, that means BglI only cut the plasmid on one site hence it is a linear DNA and the size of DNA is about 4169 bp.

C: Plasmid DNA Cut With EcoRI

It can be seen that there are two bands in lane C show that EcoRI cuts the plasmids twice. One is about 1072 bp and the other is about 5888 bp.

It is clear from lane C and lane B that 3311 bp + 1202 bp ≈ 4169 bp Hence it can be concluded that there DNA is approximately 4168 bp.

1. Analysis of D, E & F

D: Lambda DNA Uncut

Lane D represents the uncut lambda DNA hence we get only one band.