Restriction Enzymes

Because sequencing is expensive and time intensive, you can use the following method to identify individuals of different species, lineages, haplotypes, etc. (depending on which enzymes you choose). Basically, a DNA fragment is amplified by PCR, digested with a particular restriction enzyme, and the banding patterns produced by the enzyme digest(s) are visualized on an agarose gel. If you choose the right enzyme, different haplotypes will have different banding patterns.

1. There are many programs that will determine the appropriate restriction enzymes for your question. We use DNA Strider (by C. Marck). Input known DNA sequences into a restriction enzyme digestion program to determine which enzyme(s) will differentially cut your haplotypes.

2. Amplify by PCR the desired segment of DNA. You generally will only use 14 ul of the PCR product, so you can get by with a 20-25 ul PCR reaction.

3. Run 5 ul PCR reaction on a 1.5% agarose gel to ascertain success of amplification.

4. Prepare a master mix to include: 1 ul enzyme buffer (e.g. 10X NEBuffer2) + 2 units restriction enzyme (=0.25 ul of
Alu I but the number of ul will vary from enzyme to enzyme - look on the enzyme tube itself for information) per
sample. This is a miniscule amount of enzyme but diluting the enzyme in buffer for long term storage can dramatically decrease enzymatic activity over time. Just use accurate pipetters or add a little extra buffer and enzyme to your master mix to make sure you have enough mix for all of your samples. If BSA is included with enzyme, add 0.1 ul BSA (10 mg/mL) per sample to master mix.

5. Put appropriate amount of master mix into each tube and add 9.0 ul PCR product. Place in 37 C incubator for 2
1/2-3 hours.

6. Run 6 ul of digested DNA through a 1.5% agarose gel stained with ethidium bromide. You may want to run a size
standard to size bands and/or a positive control to make sure the digestion worked.

Example Form

Date:
Restriction Enzyme:
Samples:

Nla III

Master Mix:
1.0 *l 10X NEBuffer 4 X = *l Buffer
0.1 *l BSA (10 mg/mL) X = *l BSA
0.2 *l Nla III (2 units) X = *l Nla III
1.3 *l master mix per reaction

+9.0 *l PCR product (Marten 37 & MVZ 14)

Product sizes:
americana group - 41 541 153 46
caurina group - 41 200 341 199

Alu I

Master Mix:
1.0 *l 10X NEBuffer 2 X = *l Buffer
0.25 *l Alu I (2 units) X = *l Alu I
1.25 *l master mix per reaction