Methodology
1. Sampling of the material into various study groups and control group.
2. Creating environmental conditions to mimic a forensic scene.
3. Deriving of pulp & storage of samples.
4. Isolation of DNA from pulp using phenol extraction.
5. DNA quantification and quality assessment using nano spectrophotometry.
6. Obtaining primers & its design.
7. Polymerase Chain Reaction & procedure.
8. Result analysis using Gel Electrophoresis.
9. Statistical data analysis
1) Sampling:
Extracted human teeth were obtained from the Department of Oral and Maxillofacial Surgery, Vyas Dental College & Hospital. The present study was conducted by grouping the samples subjected to various environmental conditions and into Control.
Study Group
I. Freshly …show more content…
The semisolid matrix consists of a gel made from agarose.
Agarose is a polysaccharide consisting of a linear polymer (repeating units) of D-galactose and 3, 6-anhydro L-galactose.
Basic structure of Agarose
Commercially, agarose is extracted from seaweed and purified for use in electrophoresis. Movement of molecules through an agarose gel not only depends on the size and charge of molecules but also on the pore sizes present in the agarose gel. At neutral pH, DNA, RNA, and proteins migrate toward the anode (positive electrode) when an electric field is applied across the gel.
Agarose Gel Preparation
Reagents:
1 X TAE Buffer, Agarose 0.8 % Gel, (Ethidium bromide solution (10 mg/ml in water).
TAE buffer is a buffer solution containing a mixture of Tris base, acetic acid and EDTA)
1 X TAE buffer is prepared by diluting stock solution of TAE buffer which is at a concentration of 50X , by diluting 49 ml of triple distilled water and 1 ml of 50 X TAE buffer.
Gel Preparation
To prepare 50 ml of 0.8% agarose solution in TAE …show more content…
The contents are mixed well and pipetted into the sample wells formed by the combs.
The lid on the gel box was placed and the power leads on the electrophoresis apparatus were connected and an electric current of 60 volts is applied for a maximum of 2 hours or till a visible movement of the dye front was seen migrating at least half the length of the gel.
Ethidium bromide is used to stain nucleic acid and can be incorporated into agarose gels prior to gel pouring. Nucleic acid stained with ethidium bromide can be visualized under UV light. As and when a positive finding of DNA is obtained, it was quantified using a UV Spectrophotometer and its purity was checked (for each sample) to be used in Polymerase chain reaction based amplification.
5. DNA quantification and quality assessment using UV spectrophotometry.
The procedure was performed using 200 ml silica cuvettes, into which 20 μl of DNA sample was added and the rest of the volume i.e. 180 ml was made up by adding TE buffer. Along with each sample a cuvette with 200 μl of TE buffer was used as a ‘blank’ or control which gives an optical density of 0 hence served to distinguish and assess the
1. Sampling of the material into various study groups and control group.
2. Creating environmental conditions to mimic a forensic scene.
3. Deriving of pulp & storage of samples.
4. Isolation of DNA from pulp using phenol extraction.
5. DNA quantification and quality assessment using nano spectrophotometry.
6. Obtaining primers & its design.
7. Polymerase Chain Reaction & procedure.
8. Result analysis using Gel Electrophoresis.
9. Statistical data analysis
1) Sampling:
Extracted human teeth were obtained from the Department of Oral and Maxillofacial Surgery, Vyas Dental College & Hospital. The present study was conducted by grouping the samples subjected to various environmental conditions and into Control.
Study Group
I. Freshly …show more content…
The semisolid matrix consists of a gel made from agarose.
Agarose is a polysaccharide consisting of a linear polymer (repeating units) of D-galactose and 3, 6-anhydro L-galactose.
Basic structure of Agarose
Commercially, agarose is extracted from seaweed and purified for use in electrophoresis. Movement of molecules through an agarose gel not only depends on the size and charge of molecules but also on the pore sizes present in the agarose gel. At neutral pH, DNA, RNA, and proteins migrate toward the anode (positive electrode) when an electric field is applied across the gel.
Agarose Gel Preparation
Reagents:
1 X TAE Buffer, Agarose 0.8 % Gel, (Ethidium bromide solution (10 mg/ml in water).
TAE buffer is a buffer solution containing a mixture of Tris base, acetic acid and EDTA)
1 X TAE buffer is prepared by diluting stock solution of TAE buffer which is at a concentration of 50X , by diluting 49 ml of triple distilled water and 1 ml of 50 X TAE buffer.
Gel Preparation
To prepare 50 ml of 0.8% agarose solution in TAE …show more content…
The contents are mixed well and pipetted into the sample wells formed by the combs.
The lid on the gel box was placed and the power leads on the electrophoresis apparatus were connected and an electric current of 60 volts is applied for a maximum of 2 hours or till a visible movement of the dye front was seen migrating at least half the length of the gel.
Ethidium bromide is used to stain nucleic acid and can be incorporated into agarose gels prior to gel pouring. Nucleic acid stained with ethidium bromide can be visualized under UV light. As and when a positive finding of DNA is obtained, it was quantified using a UV Spectrophotometer and its purity was checked (for each sample) to be used in Polymerase chain reaction based amplification.
5. DNA quantification and quality assessment using UV spectrophotometry.
The procedure was performed using 200 ml silica cuvettes, into which 20 μl of DNA sample was added and the rest of the volume i.e. 180 ml was made up by adding TE buffer. Along with each sample a cuvette with 200 μl of TE buffer was used as a ‘blank’ or control which gives an optical density of 0 hence served to distinguish and assess the