Notes
Tuesday, April 22, 2025 6:55 PM
Maxam-Gilbert method of DNA Sequencing
• It is a method by which the sequence of a DNA fragment is identified by using chemicals, thats cut DNA at specific
points.
• Also called Chemical degradation method of DNA sequencing.
• Developed by developed by Allan Maxam and Walter Gilbert in 1976–1977
Procedure
E.g it is a fragment of double stranded DNA, and you do not its sequence:
• Step :1 As the sequence of both strands are unknown, but if we find out the sequence of one strand, we would get to
know sequence of other one also. At first, the double stranded fragment is separeted into two single strands by
applying high Temperature or high PH.
• Step :2 Run the single stranded fragments on gel. As lighter fragment band will move further than the heavy fragmen
band. How will we know which one is the lighter band? The band having larger number of purines(A,G) would be
heavier.
• Step :3 Take one of fragment band from the gel. Remove the Phosphate at 5′ end and incoporate Radioactive
Phosphate 32- PO4 enztmatically.
• Step 4: Radioactive Labelling • Now put all the radioactively labelled fragments in four tubes. 1 2 3 4
• Step 5: Chemical Degradation
Tube 1 : Increase Temperature and PH(by adding NAOH), that would cause fragments to break down. Dimethyl sulfat
will be added that would make cuts at Adenine and Guanine positions.
Tube 2: Dimethyl sulfate and dilute HCL will be added that would cuts the fragment at Adenine position
Tube 3: Reagents Hydrazine and Piperidine are added that would cuts the fragment at position Cytocine and
Thyamine.
Tube 4: In the last tube, Hydrazine, Piperdine and NACL is added that would cuts the fragment at Cytocine position.
•
Mechanism of DMS with High Temperature & NaOH
unit 3 Page 1
, Mechanism of DMS with High Temperature & NaOH
Step 1: Methylation by DMS
• DMS methylates the N7 position of guanine (G) and, to a lesser extent, adenine (A).
• This methylation weakens the glycosidic bond, making the base prone to loss.
Step 2: Depurination (High Temperature & NaOH)
• High temperature (≥90°C) and NaOH (alkaline conditions) promote depurination,
causing the loss of the methylated guanine or adenine from the DNA backbone.
• This results in an abasic (AP) site, where a purine is missing.
Step 3: Strand Cleavage
• Under alkaline conditions, the phosphodiester backbone is unstable at AP sites.
• This causes strand cleavage, leading to DNA fragmentation at the guanine and adenine
positions.
• Step 6: Gel electrophoresis - All of the fragments from each four tubes are pour in Gel. Four wells will be make on Ge
in 1st well, fragments from 1st tube is pour, in 2nd well fragments from 2nd tubes and so on. Fragments would
separate on Gel according to size. Smaller fragments would move farther than larger fragments. After placing
radioactive film on top of gel, radioactive labelled fragments would emit a spot at their position.
unit 3 Page 2
Tuesday, April 22, 2025 6:55 PM
Maxam-Gilbert method of DNA Sequencing
• It is a method by which the sequence of a DNA fragment is identified by using chemicals, thats cut DNA at specific
points.
• Also called Chemical degradation method of DNA sequencing.
• Developed by developed by Allan Maxam and Walter Gilbert in 1976–1977
Procedure
E.g it is a fragment of double stranded DNA, and you do not its sequence:
• Step :1 As the sequence of both strands are unknown, but if we find out the sequence of one strand, we would get to
know sequence of other one also. At first, the double stranded fragment is separeted into two single strands by
applying high Temperature or high PH.
• Step :2 Run the single stranded fragments on gel. As lighter fragment band will move further than the heavy fragmen
band. How will we know which one is the lighter band? The band having larger number of purines(A,G) would be
heavier.
• Step :3 Take one of fragment band from the gel. Remove the Phosphate at 5′ end and incoporate Radioactive
Phosphate 32- PO4 enztmatically.
• Step 4: Radioactive Labelling • Now put all the radioactively labelled fragments in four tubes. 1 2 3 4
• Step 5: Chemical Degradation
Tube 1 : Increase Temperature and PH(by adding NAOH), that would cause fragments to break down. Dimethyl sulfat
will be added that would make cuts at Adenine and Guanine positions.
Tube 2: Dimethyl sulfate and dilute HCL will be added that would cuts the fragment at Adenine position
Tube 3: Reagents Hydrazine and Piperidine are added that would cuts the fragment at position Cytocine and
Thyamine.
Tube 4: In the last tube, Hydrazine, Piperdine and NACL is added that would cuts the fragment at Cytocine position.
•
Mechanism of DMS with High Temperature & NaOH
unit 3 Page 1
, Mechanism of DMS with High Temperature & NaOH
Step 1: Methylation by DMS
• DMS methylates the N7 position of guanine (G) and, to a lesser extent, adenine (A).
• This methylation weakens the glycosidic bond, making the base prone to loss.
Step 2: Depurination (High Temperature & NaOH)
• High temperature (≥90°C) and NaOH (alkaline conditions) promote depurination,
causing the loss of the methylated guanine or adenine from the DNA backbone.
• This results in an abasic (AP) site, where a purine is missing.
Step 3: Strand Cleavage
• Under alkaline conditions, the phosphodiester backbone is unstable at AP sites.
• This causes strand cleavage, leading to DNA fragmentation at the guanine and adenine
positions.
• Step 6: Gel electrophoresis - All of the fragments from each four tubes are pour in Gel. Four wells will be make on Ge
in 1st well, fragments from 1st tube is pour, in 2nd well fragments from 2nd tubes and so on. Fragments would
separate on Gel according to size. Smaller fragments would move farther than larger fragments. After placing
radioactive film on top of gel, radioactive labelled fragments would emit a spot at their position.
unit 3 Page 2
