The purpose of sinister mutation testing is mainly for figuring out the molecular mechanisms of human hereditary diseases in order to effectively target these diseases for prevention, diagnosis, and treatment, but more often it is used to analyze the associations between genotypes and phenotypes of different organisms. This experiment comes from the next volume of the Molecular Cloning Laboratory Guide (3rd edition) by [American] J. Sambrook D.W. Russell.
Operation method
Single-strand conformational polymorphism and heteroduplex analysis methods for mutation detection experiments
Materials and Instruments
Amplification buffers dNTP Mixed solution Formamide Sampling buffer Sucrose gel buffer TBE Electrophoresis buffer Restriction endonucleases Heat-stabilized DNA polymerase Human genomic DNA Forward and reverse primers [a-32P]dCTP Acrylamide Bis-acrylamide Ammonium persulphate Glycerol TEMED Move makings For more product details, please visit Aladdin Scientific website.
Automatic Pipette Tips with Barrier Device Boiling Bath Electrophoresis Plates, Broken Glass and 0.4 mm Spacers Gel Drying Equipment Glass Capillaries Hamilton Syringes Microcentrifuge Tubes Adjustable Pipettes Thermal Cyclers Water Baths Whatman3 MM Filter Paper
Buffers and solutions
Refer to Appendix 1 for the composition of storage solutions, buffers and reagents.
Dilute the storage solution to the appropriate concentration.
10x Amplification Buffer
Mixed dNTP solution (PCR grade) containing 4 dNTPs, each at a concentration of 1 mml/L (PH 7.0)
Formamide Sampling Buffer
Sucrose Gel Buffer (Buffer Type I)
10XTBE Electrophoresis Buffer
Polyacrylamide gel electrophoresis is performed with 1X working strength TBE (89 mmol/L Tris-borate, 2 mmol/L EDTA). 1xTBE concentration provides the necessary buffering capacity. The pH of the buffer should normally be 8.3. pH adjustments are not usually necessary, however, when using 10XTBE buffer freshly prepared in the laboratory, pH changes should be monitored.
Use the same 10XTBE storage solution for both gel and run buffers. Small changes in ionic strength and pH will produce buffer antagonism. The DNA banding pattern will be greatly distorted.
Enzymes and Buffers
Restriction endonuclease (optional)
Heat-stable DNA polymerase
Taq DNA polymerase recommended
Nucleotides and Oligonucleotides
For screening human genomic DNA for point mutations
DNA at 10ug/ml in TE (pH 7.6) buffer.
Oligonucleotide primers including forward and reverse primers (each at a concentration of 35umol/L) were dissolved in TE (pH7.6).
Radioactive material
[ a-32P ]dCTP (3000Ci/mmol, 10mCi/ml)
Gel
Acrylamide: bisacrylamide (29:1;m/V)
Many researchers use commercially available preformed acrylamide and bisacrylamide solutions (e.g., .Acrylogel BDH). However, the best resolution is obtained using the Hydrolink series of coagulant amines (Molinarif et al. 1993), commercially known as MDE (Mutation Detection Enhancement), manufactured by FMC Bioproducts (Rockland, Maine).
Amine persulfate (10% w/V)
Glycerol
Use a high purity grade of glycerin, such as ultrapure (Life Technologies). See the information section on glycerol.
TEMED (Tetramethylethylenediamine)
Many manufacturers . including Sigma and Bio-Rad, sell electrophoresis grade TEMED. TEMED is hygroscopic and should be stored in an airtight container at 4° C. The product should not be used for electrophoresis.
TEMEU serves as a co-catalyst for the polymerization of acrylamide.
Specialty Equipment
Automatic pipette tips with barrier device
Boiling water baths
Electrophoresis plates, broken glass (two 40 cmX40 cm glasses) and 0.4 mm spacers.
Gluing equipment
Glass capillary (elongated) or micropipette with gel pipette tip
Hamilton syringes or Pasteur pipettes
Microcentrifuge tubes (thin-walled 0.5 ml tubes for 闬)
Adjustable pipettes
Thermal cycler capable of programming amplification protocols
If the thermal cycler is not equipped with a heated lid, use light mineral oil or paraffin to prevent liquid evaporation from the reaction mixture during PCR.
Water bath at the temperature required for restriction endonuclease digestion
Whatman3 MM Filter Paper
Method
Amplification of DNA for screening point mutations
1. Mix the following components in order in a sterilized centrifuge tube.
1 mmol/LdNTP solution 1ul
10x Amplification Buffer 2ul
35umol/L 5'-oligonucleotide solution 1ul
35umol/L 3'-oligonucleotide solution 1ul
10uCi/ul [ a-32Pld-CTP 1ul
Human genomic DNA 10ul(100ng)
Heat-stabilized DNA polymerase 1~3 units
Add water to 20ul
During PCR [ a-32P ]dCTP is uniformly bound and labeled to the amplified DNA. Replace [ a-32P ]dCTP with a 32P-labeled oligonucleotide primer to produce end-labeled DNA.
If possible, set up two sets of DNA samples known to contain one or more bases different in the allelic free sequence and known to be distinguishable on SSCP gels as controls. An additional negative control without template DNA is required to avoid contamination.
2. If the thermal cycler does not have a heated lid device, add 1 drop (~50ul) of light mineral oil to cover the reaction mixture to prevent sample evaporation during repeated heating and cooling cycles. If using a hot start program, add a drop of paraffin oil to the tube. Place the reaction tube in the thermal cycler.
3. Perform amplification by following the denaturation, annealing, and polymerization times and temperatures listed in the table below. Refer to Program I in Chapter 8 for setting thermal cycling parameters.
The above reaction conditions are suitable for 50ul reaction volumes, 0.5 ml thin-walled tubes, and Perkin-Elmer 9600, 9700, Master Cyder (Eppendorf), or PTC100 (MJ Research) thermal cyclers. Adjustment of the above reaction conditions is required when using other types of instruments or different reaction volumes.
Preparation of SSCP gels
4. When starting the PCR reaction, prepare a 5.5% polyacrylamide gel with lxTBE buffer containing 10% (V/V) glycerol.
10XTBE gel buffer 10 ml
29:1 Acrylamide: Bisacrylamide Solution 18 ml
Amine persulfate 10% 0.5 ml
Glycerol 10 ml
Add water to 61.5 ml
Stir gently to mix the above reagents.
The above volume of solution is sufficient to fill a standard size (40 cmX40 cm) gel with a 0.4 mm spacer thickness. The volume of gel solution can be increased or decreased to prepare a gel of the desired size.
Prepare a 1XTBE gel buffer sufficient to fill the gel electrophoresis unit with the same 10XTBE storage solution.
5. Assemble and dip two 40 cmX40 cm glass electrophoresis plates with 0.4 mm spacers.
For maximum resolution of single-stranded DNA conformers, use spacers with a gel thickness equal to or less than 0.4 mm.
6. Add 100ul of TEMED solution and shake the vial gently to mix the solution and fill the gel.
The acrylamide solution polymerizes rapidly, so work quickly. See Option 8 in Chapter 12 for instructions on filling thin gels.
7. Place the polymerized gel into the electrophoresis apparatus at room temperature. Fill the electrophoresis bath with the same 1XTBE buffer used to prepare the gel.
Preparing Samples for SSCP Electrophoresis
8. (Optional) At the end of the PCR reaction, remove the reaction tube and place it on ice. If digestion with restriction enzymes is required to amplify the DNA fragments, set up the following digestion reaction.
PCR solution 5ul
Restriction buffer 4ul
Restriction endonuclease (2~50 units) 2ul
Water 29ul
Incubate for 1~2 h at a temperature suitable for restriction enzyme digestion. 
9. 1.5ul of the original PCR product (step 3) or 5ul of the restriction digested PCR product (step 8) was diluted to 20ul with Sucrose Gel Sampling Buffer. the same amount of sample was diluted to 20ul with a formamide dye mixture.
Samples diluted with formamide dye mixture will be denatured, while samples diluted with sucrose gel buffer will remain double-stranded and serve as controls.
10. Boil the formamide-containing sample for 6 min, then insert the reaction tube directly into ice.
Separation and Analysis of DNA Fragments by SSCP Gel Electrophoresis
11. Rinse the wells of the polyacrylamide gel with 1XTBE buffer using a Pasteur dropper or Hamilton syringe. Add 2ul of sample to the polyacrylamide gel electrophoresis using an elongated glass capillary or a micropipette with a gel pipette tip.
12, Electrophoresis was performed for 14 h using a voltage of 6~7 V/cm [about 250 V (and 15 mA) for a 40 cmX40 cm gel].
13, After completion of electrophoresis, separate the glass plate, transfer the gel onto 1 layer of Whatman 3 MM filter paper and vacuum dry for 30-60 min.
14. Radiographic autoradiography was performed on the dried gel, which was exposed for 4-16 h at room temperature without a sensitizing screen.
Non-denatured PCR samples (i.e., samples diluted with Sucrose Gel Sampling Buffer) migrate through the gel as double-stranded DNA. In contrast, denatured samples (i.e., samples mixed with a formamide dye mixture and boiled) migrate through the gel as a mixture of double- and single-stranded DNA. Single-stranded DNA generally migrates less in acrylamide gels than double-stranded molecules (Majwm and Gilbert 1977, 1980; Szalay et al. 1977). In cases where the two DNA complementary strands fold to form a configuration that cannot be resolved by SSCP, there is -a single-stranded band that can be detected. If the complementary strands fold into a distinguishable configuration . Both strands are detected. The PCK product of a heterozygous allele in a diploid organism produces at least four bands, two of which have the same mobility as the wild-type and two of which are specific mutants. However, there are often more than two bands, either as a result of genetic heterogeneity of the samples or because the two complementary strands of the same DNA molecule have folded in on themselves to form more than one conformation. Band patterns are quite complex and difficult to derive from DNA sequence, base composition, and length of fragments. However, there is usually a characteristic band pattern for a particular mutation.