The Maxam-Gilbert sequencing method involves chemically cleaving DNA strands at specific bases, labeling the fragments radioactively, and separating them via gel electrophoresis. The sequence can then be read from the banding pattern on X-ray film. This early chemical degradation method was developed in 1977 and provided one of the first reliable ways to determine DNA sequences, but it uses toxic chemicals and radioactivity, has technical complexity, and can only read up to 500 base pairs.

2. MAXAM GILBERT
SEQUENCING METHOD
Presented by Natasha Subhan

3. DNA SEQUENCING
 THE DNA SEQUENCING INVOLVE THE PROCESS OF FIGURING OUT THE
PRECISE ORDER OF FOUR BASES (A,T,G,C) FOUNAD IN DNA.
 SIMPLY IT IS THE PROCESS OF DETERMINING THE ORDER OF NUCLEOTIDES
IN DNA
 METHODS USED FOR DNA SEQUENCING ARE:
 MAXAM GILBER SEQUENCING METHOD
 SANGER SEQUENCING METHOD
 NEXT GENERATION DNA SEQUENCING METHOD

4. APPLICATIN OF DNA SEQUENCING:
 WITH ITS STUDY WE CAN UNDERSTAND THE SEQUENCE RESPONSIBLE
FOR ANY DISEASE.
 WE CAN DETECT ANY MUTATION.
 DNA FINGER PRINTING.
 FORENSICS.
 AGRICULTURE.

5. MAXAM GILBERT SEQUENCING METHOD
 It is a method by which the sequence of a DNA fragment is
identified by using the chemicals, that cut the DNA at specific
points .
 Also called chemical degradation method of DNA sequencing
 Develop by Allan Maxam and
Walter Gilbert in 1976 to 1977

6. PROCEDURE
 Lets suppose it is a fragment of double stranded DNA & we
don't know its sequence

7. 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 as well.
 High pH or high temperature
 At first the double stranded DNA is separated into two
single strands by applying high pH or high temperature.

9. STEP : 2
 Run the single stranded fragment on gel
 Lighter fragment will move faster than the heavy fragment
band
 How will we know which one is the lighter band ?
 The band having larger number of purines ( A, G) would be
heavier .

10. Step:3
 Take one fragment band from the gel
 Remove the phosphate at 5 prime end and incorporate
Radioactive Phosphate 32-po4 enzymatically

11. Step:4 Radioactive labelling
 Now put all the radioactive labelled fragments in four tubes .

12. Step:5 Chemical degradation
 Tube 1: increase temp and p H ( by adding NAOH) that
would cause fragments to break down.
 Dimethyl sulfate will be added that would make cuts at
Adenine and Guanine positions.
 Tube 2: dimethyl sulfate and dilute HCL will 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

13.  TUBE:4 In the last tube, Hydrazine, Piperdine and NACL is
added that would cuts the fragments Cytosine position.

14.  After chemical degradation we will get the following radioactively
labelled fragments from each tube

15. Step : 6 GEL ELECTROPHOROSIS
 All fragments from each four tubes are pour in gel
 Four pores will be make on gel
 In first pore, fragments from 1st tube is poured
 & in 2nd pore fragments from 2nd tubes and so on.
 Fragments will 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.

16. GEL ELECTROPHOROSIS:

17. ADVANTAGES & DISADVANTAGES
 ADVANTAGES:
 Directly read purified DNA
 No premature termination due to DNA sequencing , so no problem
with polymerase to synthesize DNA.
 Used sequence heterogeneous DNA as well as homopolymeric
sequences
 Used to analyse DNA protein interaction i.e footprinting
 Used to analyse epigenic modification and nucleic acid structure to
DNA.
 DISADVANTAGES:
 Not widely used.
 Use of toxic chemicals and extensive use of radioactive isotopes, highly
poisonous and unstable

18.  Cannot read more than 500bp.
 Setup is quite complex, technically complexity
 It is difficult to make Maxam Gilbert based DNA
sequencing kit.
 Read size decrease with incomplete cleavage reactions

19. Limitations :
 Gel electrophoresis is limited to700 to 900 bp, with 400 to 500
more commonly attained
 The first 15 t0 40 bp are often difficult to interpret
 Time acquire process i.e
 time spend on loading and running samples on gels
 autoradiography
 and analysis , all lower the amount of DNA that can be
sequenced