Single Strand Conformation Polymorphism (SSCP) Analysis by Nondenaturing PAGE
|Author: B.R. Yadav and D.S. Kale|
|Affiliation: Livestock Genome Analysis Laboratory (DCB Division), National Dairy Research Institute (NDRI), Karnal-132001, Haryana, India. Email: email@example.com & firstname.lastname@example.org|
|Date Added: Mon Feb 02 2009|
|Date Modified: Mon Feb 02 2009|
|Abstract: This protocol, with brief principle and procedure can be informative and useful for molecular biology researchers working on genetic polymorphism analysis of QTLs such as Leptin, STAT5A, DGAT and Butyrophilin candidate genes using SSCP technique.|
Single-strand conformation polymorphism (SSCP) technique is a simple and efficient means to detect any small alteration in PCR-amplified product. It is based on the assumption that subtle nucleic acid change affects the migration of single-stranded DNA fragment and, therefore, results in visible mobility shifts across a nondenaturing polyacrylamide gel (Orita et al., 1989). Polyacrylamide gel is used for analysis of DNA with specialized buffer systems and without urea. In nondenaturing PAGE the components used to synthesize matrix are acrylamide monomers, N, N-methylene bisacrylamide (Bis), ammonium persulphate (APS) and N,N,N’,N’-tetramethylenediamine (TEMED). Ammonium persulphate (APS) when dissolved in water generates free radicals, which activate acrylamide monomers inducing them to react with other acrylamide molecules forming long chains. These chains cross-linked with Bis.TEMED act as catalyst for gel formation because of its ability to exist in free radical form. The acrylamide and bisacrylamide is used in 49:1 ratio using autoclaved HPLC water added to make 100 ml volume. This 49:1 acrylamide-bisacrylamide solution is dissolved completely using magnetic stirrer and kept it in refrigerator till used.
Preparation of SSCP Gel
The percentage of gels used for SSCP analysis varies from 8-20 percent but most of the primers were optimized with good results in 12% PAGE solution.
Composition of 12% nondenaturing PAGE Solution (For 100 ml)
Component Volume 50% Acrylamide-bisacrylamide 24 ml 10 X TBE 5 ml Glycerol 10 ml Autoclaved HPLC water 61 ml
The gel mixture is kept dissolved completely and stored at -20°C till it is used.
The PCR-SSCP procedure includes following steps viz; PCR amplification of the gene fragments, resolution in nondenaturing PAGE and visualization using silver staining. PCR conditions are optimised for PCR-SSCP by testing a number of variables such as concentration of DNA, Taq polymerase, dNTPs, MgCl2 and temperature profile. The PCR amplification protocol for all the SSCP primers used is same except the annealing temperature, which varies between primers.
- The Single Strand Conformation Polymorphism analysis of amplified gene fragments is carried out using Bio-Rad Protein II xi Cell vertical gel electrophoresis unit (Bio-Rad laboratories). The two glass plates are washed thoroughly using tap water with detergent and rinsed initially under running tap water till no remains of detergent are left. The plates are wiped two times with tissue paper soaked in distilled water first, 70 percent alcohol and then air-dried. The similar thorough cleaning treatment is given to spacers and comb to ensure proper alignment of 20 cm glass plates.
- The gel sandwich is assembled on a clean surface laying down the long rectangular plate first, then two spacers of equal thickness along the long edges of plate and the short plate is placed on the rectangular plate. The two glass plates with spacers between them are fitted well with proper alignment by tightening the bulldog clamps. The sandwiched gel plates are fitted in the stand with screw clamps. The cleaned comb (20 wells) is inserted from the topside of the gel sandwich and immediately bulldog clamps are applied over the plates containing comb to create sharp wells.
- The bottom side of the gel sandwich is sealed using 10 ml of 12% gel mix. The gel sandwich is kept in slanting position and the solution mixed with 50 µl APS and 20 µl TEMED is injected between the two glass plates using syringe fitted with 10 µl tip and allowed to polymerize for 10 minutes.
- After polymerisation the assembled gel sandwich is placed in alignment slot of casting stand. The 12% native PAGE gel mix (25 ml) is prepared by adding APS (100 ul) and TEMED (40 µl) at a time and mixed well. This gel mix is filled from upper side of gel sandwich using syringe smoothly without any bubble and clamps are immediately applied over the comb to ensure sharp wells. The gel is kept undisturbed at least 45 minutes for polymerisation.
- After polymerisation the comb is removed and wells are flushed with 0.5X buffer. The gel sandwich is placed in electrophoresis tank with notched plate facing towards the buffer reservoir. The reservoir of the electrophoresis tank is filled with 0.5X TBE and the gel is given pre-run at 200 volts at constant temperature for minimum 45 minutes. Ice cooled water circulation with electric pump is applied to central cooling core of assembly to maintain constant temperature.
- About 4 µl PCR product and 12 µl of a formamide dye is prepared in PCR tube and denatured at 95°C for 10 minutes in the Biometra PCR machine. After denaturation the samples are immediately kept in ice-chilled box and kept in -20°C deep freeze for 10 minutes.
- After completion of pre-run the wells are flushed again using buffer. The samples are loaded on a nondenaturing 12% acry1amide: bis-acrylamide (49:l) gel with gel loading tip and immediately electrophoresis is performed in 0.5 X Tris borate (pH 8.3)-EDTA buffer at 10-12.5 volts/cm for 3-24 hr at room temperature depending on the optimised conditions for each primer.
- After completion of the electrophoresis for required time the glass plates are removed from the assembly. There after gel is subjected to silver staining to visualize SSCP band patterns.
- In order to stain the gel it is immersed in a tray of appropriate size filled with 10% acetic acid (500ml) for at least 30 minutes for fixing DNA bands in gel so as to prevent diffusion of the DNA bands (Care has to be taken so that gel remains dipped well in solution). The gel is agitated slowly for 30 minutes or until the tracking dye is no longer visible.
- The acetic acid is decanted and 500 ml of distilled water is poured in the tray and rinsed thoroughly by placing the tray on oscillatory automatic shaker for 20 minutes.
- Meanwhile 500 ml of 0.1% silver nitrate solution is prepared in amber color bottle and 750 µl of 37% formaldehyde is added and mixed. Distilled water is gently decanted from tray. The gel is stained for 45 minutes in silver nitrate solution with constant shaking in a dark room or covering the tray with black cloth.
- Then the gel is rinsed briefly for 25 seconds in distilled water.
- Working quickly distilled water is decanted from the tray. The freshly prepared and chilled 3% sodium carbonate solution (3% Na2CO3 and 750µl of 37% formaldehyde + 1% sodium thiosulfate) is transferred to the tray. The gel is kept immersed until bands get developed sharply.
- The gel is given 10 percent acetic acid (stop solution) treatment for 10 minutes. Then 500 ml distilled water is added to tray.
- The gel is transferred gently on the transparency; excess water is soaked with tissue paper, and air dried for half an hour.
The silver stained gel is kept on transilluminator and SSCP variants are recorded. Then gel is labeled and scanned for computer image analysis and documentation.