Mutations are changes in DNA sequence and are the cause of genetic variation and drive evolution. The Surveyor® Mutation Detection Kit for Standard Gel Elecrophoresis provides a method to scan mutations and polymorphisms in DNA. It is based on: 1) PCR amplification of reference and test samples 2) hybridization of these two PCR products to create a heteroduplex with mismatch due to insertion/deletion or base-substitution, 3) digestion with a mismatch-specific DNA endonuclease that cuts both strands of the DNA heteroduplex on the 3’- side of the mismatch site 4) analyses of obtained fragments with agarose or polyacrylamide gel electrophoresis.
The first step is critical for the successful digestion with Surveyor Nuclease, so it is important follow a few recommendations. A proofreading DNA polymerase for PCR amplification is not provided. I have used Optimase Polymerase (Transgenomic) because I had PCR products < 2,500 bp. In order to be certain that the polymerase you are using is compatible with the kit, contact Transgenomic for the recommended DNA polymerases. If you use Optimase Polymerase, you will need to ensure that you have is 100 ng of genomic DNA. Primer placement is also crucial. If you have a small amplified DNA fragment (<1,000 bp), place primers at least 50 bp outside the region of interest to ensure that the cleavage products are at least 70 bp long; otherwise, it will not be possible to see the band on an agarose gel. If the DNA concentration after amplification is less than 25 ng/ìl, you will need to concentrate by ethanol precipitation.
The use of a heated-lid thermocycler is recommended for DNA hybridization. Mix equal amounts of test and reference PCR products in a 0.2 ml tube and ensure that the final volume is at least 10 ìl so that each tube contains at least 200 ng total DNA. Test and reference samples are also self-hybridized and used as digestion controls. The thermocycler program is: 95°C for 2 min; 95°C to 85°C with a decrement of 2°C/s; 85°C to 25°C with a decrement of 0.1°C/s and 4°C hold.
For each digestion, you have to use a nuclease-free 0.2 ml tube and add 200 to 400 ng of hybridized DNA, 0.5 to 2 ìl of Surveyor® Enhancer S and Nuclease S (ratio 1:1). Mix gently and incubate at 42°C for 20 min. Add 1/10 volume of Stop Solution and mix. Store the digestion products at -20°C or analyze them immediately. For small digestion products, I suggest using Agarose MS-6 Metagel (Conda) and running in 1x TBE. For each mutation you will see two bands.
In our lab, we have tested the hypothesis of positive selection on different genes of Botrytis cinerea and we have used the Surveyor® Mutation Detection Kit for Standard Gel Elecrophoresis for preliminary analysis to detect different or identical sequences among 32 strains of this fungus. In this way, we have sequenced only the polymorphic sequences. In conclusion, we can say that this kit is a valid tool to scan for a few mutations. If you have a lot and the mutations are located near polymorphic sites, you will see them on the agarose gel as a single band.
When you use the kit for the first time, it is not so easy, because you first have to use the plasmid provided with the kit to test whether the chosen polymerase works. You also have to optimize the quantity of DNA for hybridization and then digestion. Also, you can spend a lot of time in quantification at the spectrophotometer if you have many samples. Nevertheless, once you have the parameters set, it works consistently well.