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Objectives:Objectives:
• Introduce the students to digest genomic DNA by restriction endonucleases.
• Observe the results of digestion on agarose gel electrophoresis.
Introduction:• Restriction enzymes (also known as
restriction endonucleases) found in bacteria (and harvested from them for use) and cut DNA up at specific sequences in the genome (blunt or sticky ends).
For example, the commonly used restriction
endonuclease EcoRI recognizes every point in DNA with
the sequence GAATTC, and cuts at the point between
the Guanine and Adenine.
• Whereas SmaI restriction enzyme cleavage produces "blunt" ends:
Nomenclature
Since their discovery in the 1970s, more than 100 different restriction enzymes have been identified in different bacteria.
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, the name of the EcoRI restriction enzyme was derived as shown in the box.
AbbreviationMeaning
Description
EEscherichia
genus
cocoli species
RRY13 strain
IFirst
identified
order of identification
in the bacterium
Derivation of the EcoRI
name
• Restriction enzymes are part of a bacteria's ''immune'' system.
• These are enzymes that cut DNA at specific sites (typically a four or a 6 base-pair sequence).
• Bacterial DNA is modified to be protected by methylation while foreign DNA, such as incoming viruses, are not.
Thus, we can take a long piece of DNA and cut it with a restriction enzyme, generating numerous fragments. Even a single-base change will destroy a restriction enzyme target site. Likewise, even if a site is the same in two molecules, the length of DAN sequence between them may change. Thus if two DNA molecules differ in sequence, they likely have different lengths for the fragments produced following treatment with restriction enzymes.
• Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain, in contrast to exonucleases, which cleave phosphodiester bonds at the end of a polynucleotide chain.
• Typically, a restriction site will be a palindromic sequence four to six nucleotides long. Most restriction endonucleases cleave the DNA strand unevenly, leaving complementary single-stranded ends. These ends can reconnect through hybridization and are termed "sticky ends."
DNAs with the ECOR1 specific DNA sequence at different places will be cut into fragments of different lengths.
DNAs of different sizes can be separated on agarose gels.
Procedure :
1. Warm your TE/DNA mixture at 55C for 15 -30 minutes. Pipet or light vortex to resuspend.
2. Prepare an enzyme digest of DNA by adding the required components to a clean microtube in the following order:1. 14 µl H2O2. 2 µl appropriate enzyme buffer (10X)3. 3 µl DNA4. 1 µl of enzyme (use BamHI, ClaI, EcoR, HaeIII, or HindIII)
3. Flick the tube to mix well and spin for 5 sec in the microfuge to bring all the components to the bottom.
4. Incubate at 37ºC in a water bath for two hours or overnight.
5. Run a gel (1% agarose gel as describe in Lab. 3 ) or freeze the samples until you have time to run the gel.
6. Record your observation.
• Different restriction enzymes that recognize the same sequence are known as neoschizomers. These often cleave in different locales of the sequence.
• Different enzymes that recognize and cleave in the same location are known as isoschizomers.
