DNA sequencing.pptx

1. DNA SEQUENCING

2. SANGER SEQUENCING:
THE CHAIN TERMINATION METHOD
 Sanger sequencing, also known as the “chain termination method,” was developed by
the English biochemist Frederick Sanger and his colleagues in 1977. This method is
designed for determining the sequence of nucleotide bases in a piece of DNA
(commonly less than 1,000 bp in length).
 Sanger sequencing was used in the Human Genome Project to determine the
sequences of relatively small fragments of human DNA (900 bp or less). These
fragments were used to assemble larger DNA fragments and, eventually, entire
chromosomes.

3. Sanger sequencing is a DNA sequencing method in which target DNA is denatured and
annealed to an oligonucleotide primer, which is then extended by DNA polymerase
using a mixture of deoxynucleotide triphosphates (normal dNTPs) and chain-terminating
dideoxynucleotide triphosphates (ddNTPs).
 ddNTPs lack the 3’ OH group to which the next dNTP of the growing DNA chain is
added. Without the 3’ OH, no more nucleotides can be added, and DNA polymerase
falls off.
 The resulting newly synthesized DNA chains will be a mixture of lengths, depending
on how long the chain was when a ddNTP was randomly incorporated.

4. How it Works:
 First, anneal the primer to the DNA template (must be single stranded):
5’ -GAATGTCCTTTCTCTAAG 3'-
GGAGACTTACAGGAAAGAGATTCAGGATTCATACCATGAAGATCAAG-5'
 Then split the sample into four aliquots including the following nucleotides:
"G" tube: All four dNTPs plus ddGTP (low concentration)
"A" tube: All four dNTPs plus ddATP
"T" tube: All four dNTPs plus ddTTP
"C" tube: All four dNTPs plus ddCTP

5. When a DNA polymerase is added to the tubes, the synthetic reaction proceeds until, by
chance, a dideoxynucleotide is incorporated instead of a deoxynucleotide. This is a
"chain termination" event, because there is a 3' H instead of a 3' OH group, required for
phosphodiester bond formation; therefore, when DNA polymerase incorporates a ddNTP
at random, extension ceases.
The result of chain-termination PCR is millions to billions of oligonucleotide copies of
the DNA sequence of interest, terminated at a random length.

6. In the second step, the chain-terminated oligonucleotides are separated by size via gel
electrophoresis. In gel electrophoresis, DNA samples are loaded into one end of a gel
matrix, and an electric current is applied; DNA is negatively charged, so the
oligonucleotides will be pulled toward the positive electrode on the opposite side of
the gel.
Because all DNA fragments have the same charge per unit of mass, the speed at which
the oligonucleotides move will be determined only by size. The smaller a fragment is,
the less friction it will experience as it moves through the gel, and the faster it will
move.
In result, the oligonucleotides will be arranged from smallest to largest, reading the gel
from bottom to top.

7. The last step simply involves reading the gel to determine the sequence of the input
DNA.
Because DNA polymerase only synthesizes DNA in the 5’ to 3’ direction starting at
a provided primer, each terminal ddNTP will correspond to a specific nucleotide in
the original sequence (e.g., the shortest fragment must terminate at the first
nucleotide from the 5’ end, the second-shortest fragment must terminate at the
second nucleotide from the 5’ end, etc.)
Therefore, by reading the gel bands from smallest to largest, we can determine the 5’
to 3’ sequence of the original DNA strand.

10. MAXAM-GILBERT METHOD / CHEMICAL DEGRADATION
METHOD
In 1976–1977, Allan Maxam and Walter Gilbert developed a DNA sequencing method based on
chemical modification of DNA and subsequent cleavage at specific bases.
DNA extraction is the very first step. After that, the DNA is denatured using the heat
denaturation method and single-stranded DNA is generated.
The phosphate (5’ P) end of the DNA is removed and labeled by the radiolabeled P32.
The enzyme named phosphatase removes the phosphate from the DNA and
simultaneously, the kinase adds the 32P to the 5’ end of it.

11.  A two-step catalytic process involves piperidine and two chemicals that selectively
attack purines and pyrimidines. Purines will react with dimethyl sulfate and
pyrimidines will react with hydrazine in such a way as to break the glycoside bond
between the ribose sugar and the base displacing the base.
 Piperidine will then catalyze phosphodiester bond cleavage where the base has been
displaced.
 Moreover, dimethyl sulfate and piperidine alone will selectively cleave guanine
nucleotides but dimethyl sulfate and piperidine in formic acid will cleave both
adenine and guanine nucleotide.
 Similarly, hydrazine and piperidine will cleave both thymine and cytosine nucleotides
whereas hydrazine and piperidine in 1.5M NaCl will only cleave cytosine nucleotides.

12. The labeled substrate (DNA) would be subjected to four separate cleavage reactions,
each of which would create a population of labeled cleavage products ending in known
nucleotides.
The reactions would be loaded on high percentage polyacrylamide gels and the
fragments resolved by electrophoresis.
The gel would then be transferred to a light-proof X-ray film cassette, a piece of X-ray
film placed over the gel, and the cassette placed in a freezer for several days.
Wherever a labeled fragment stopped on the gel the radioactive tag would expose the
film due to particle decay (autoradiography).

13. Since electrophoresis, whether in acrylamide or an agarose matrix, will resolve nucleic
acid fragments in the inverse order of length, that is, smaller fragments will run faster in
the gel matrix than larger fragments, the dark autoradiographic bands on the film will
represent the 5’→3’ DNA sequence when read from bottom to top.
The process of base calling would involve interpreting the banding pattern relative to the
four chemical reactions. For example, a band in the lanes corresponding to the C only
and the C + T reactions would be called a C. If the band was present in the C + T
reaction lane but not in the C only reaction lane it would be called a T. The same
decision process would obtain for the G only and the G + A reaction lanes.