Research Methods 2.6 Restriction Enzyme Digest

what does restriction enzyme digestion do

will cut circular plasmids into smaller linear pieces to confimer insert of PCR product into pJET vector by visualization on an agarose gel to analyze for correct sizes and indicate insertion of PCR products

what is a restriction enzyme

it is sequence specific meaning that it recognizes specific sequences, cleaves DNA with blunt ends or sticky ends, and often works at 37C, and most of them can be heat inactivated

uses of a restriction enzyme digest

1. in cloning - insertion of genes into plasmid vectors

2. protein experssion experiments - ligation of cDNA into a plasmid vector

3. allele recognition based on single nucleotide polymorphisms (SNPs) - distinguishing gene alleles based on SNPs

4. DNA sequencing

what are other things REs can be used in

1. buffers

2. double digests

3. recognition site vs cleavage site

types of restriction enzymes

some enzymes utilize ATP while others dont, and some enzymes want divalence and variety

what kind of enzyme is the RE enzyme

naturally occuring enzyme that cut both strands of dsDNA, was originally a defense mechanism against invading viruses called bacteriophages that inject DNA into bacteria. bacteria developed enzymes that recognize specific sequences within phage DNA and then cut the DNA at that site preventing the phage from destroying the bacteria.

endonucleases

enzymes used in cloning that cut at an interior location in the DNA molecule

type II restriction endonucleases

recognize and bind to specific sequences in the DNA (called a restriction site) and cuts the DNA molecule at only that sequence

how many BP are most restriction sites

6

how do restriction enzynes cut DNA

in 1 of 2 ways: both strands and staggered points

both strands

cut through both strands between 2 adjacent base pairs leaving blunt ends on either side of the cut

staggered points

cut at staggered points on the 2 DNA strands leaving a single stranded overhand on each new DNA molecule (sticky/cohesive ends). this is very useful in cloning, BgII produces sticky ends with a 5’ overhang

optimal conditions for restriction enzyme

• optimal growth temperature

• Mg2+

• sensitivity

• pH

• inactivation

optimal growth temperature

for the bacteria in which it was found, most around 37C

Mg2+

most REs need Mg2+ for activity, RE buffers also contain NaCl

sensitivity

some enzymes are sensitive to the presence of other ions like Mn2+ and may have reduced or no activity if present

pH

most enzymes are most active at pH slightly above neutral (7.2-8.0)

inactivation

after DNA is cut with RE, RE must be inactivated so it does not interfere with subsequent experiments. some are heat inactivated at 65C for 25 min or 80C, but BgII cannot be inactivated.

determining size of insert

RE digestion is followed by gel electrophoresis, and the gene or portion of the gene that was inserted into the plasmid vector should be known. digesting mini prep DNA with BgII allows insert to be cut out of vector.

gel

2 bands present in digested with one being the size of the insert, and 1 the size of the vector. if there are more than 2 band, it is a BgII site or a mixed culture

circular plasmid DNA

supercoiled DNA that is tightly wound and compacted, moves faster than linear DNA and appears smalled than plasmids cut by RE

nicked DNA

still circular but one strand of double helix has been cut, DNA no longer supercoiled, travels like linear DNA

pGAP

positive control

incomplete digestion

1 faint band one present

no band

no insert

low band

primer dimer

large band

is okay because genes can be different sizes but not too large or that is nonspecific amplification

undigested DNA

supercoiled and compacted, runs faster, appears smaller than digested.