Learning Outcomes

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At the end of this lecture, students will be able to: define sequencing Compare between the enzymatic and chemical sequencing method Explain how chemical sequencing method is carried out Describe the differences of automated sequencing method compared to the conventional sanger’s dideoxy method Lists applications of sequencing

Sequencing of DNA
 DNA sequencing is the determination of the precise

sequence of nucleotides in a sample of DNA
Sequencing is principally classified by two methods:  Enzymatic sequencing
(or named after its developers 'Sanger-Coulson- Sequencing')

 Chemical sequencing method
(or named 'Maxam-Gilbert-Sequencing' after its developers)

Maxam & Gilbert's method (1976)
-chemical cleavage method using double stranded DNA -involves 2 steps catalytic process  2 chemicals that selectively attacks purines or pirimidines (step 1)  Piperidine will catalyze phosphodiester bond cleavage where the base has been displaced (step 2)

Step 1:Displacing the base step
During this step glycoside bond between the ribose sugar and base is broken Purines (A & G) will react with dimethyl suphate Pyrimidines (C & T) will react with hydrazine

Chemicals used Dimethyl sulphate and Piperidine

Base specificity Guanine

Dimethyl sulphate and Guanine and Adenine Piperidine in Formic acid Hydrazine and Piperidine Thymine and Cytosine

Hydrazine and Piperidine in 1.5M NaCl

Cytosine

 Difficult chemical cleavage method and is no longer used

Sanger’s dideoxy method (1977)
This technique utilizes 2',3'-dideoxynucleotide triphospates (ddNTPs), molecules that differ from deoxynucleotides by having a hydrogen atom attached to the 3' carbon rather than an OH group. These molecules terminate DNA chain elongation because they cannot form a phosphodiester bond with the next deoxynucleotide.

Deoxyribose vs Dideoxyribose

DNA polymer

3’ OH is necessary for polymerisation

Sanger’s dideoxy method
During the enzymatic sequencing, the DNA to be sequenced is multiplied with a synthesisreaction. This method takes part in two steps: the labelling-reaction in order to produce a labelled source sample

the termination-reaction where the synthesis of the fragments produced in the first reaction is ended

Sanger’s dideoxy method
the DNA segment of interest is heated to disassociate the strands To sequence a DNA fragment each of 4 reaction tubes is prepared with ssDNA template, DNA polymerase, 32 P labeled primer. Each tube receives a small amount of a different ddNTP, together with the four normal dNTPs. A ddNTP will be incorporated randomly, at different sites in different syntheses in the reaction tube. in any given tube various truncated chain lengths will be produced, each corresponding to the point at which the respective ddNTP for that tube was incorporated and terminated chain growth. Because incorporation is random, all possible truncated fragments will be produced, corresponding to all the various positions of that particular base. Laws of probability will determine the sequence length. Sample in each tube loaded onto adjacent lanes on a sequencing gel, electrophoresed, then exposed to film to produce a autoradiogram. Base sequence determined by scanning up the gel.

Sanger’s dideoxy method

Autorad of a sequencing gel from dideoxy method

Autoradiograph is used to identify the positions occupied by the fragments.

Automated DNA sequencing method (1990s)
Based on Sanger’s dideoxy method for sequencing Able to use robotic and computer systems Also known as cycle sequencing Can obtain millions of bases of sequence per day This template DNA is supplied with a mixture of all four normal (deoxy) nucleotides in ample quantities – dATP – dGTP – dCTP – dTTP a mixture of all four dideoxynucleotides, each present in limiting quantities and each labeled with a "tag" that fluoresces a different color: – ddATP – ddGTP – ddCTP – ddTTP DNA polymerase I with buffer Because all four normal nucleotides are present, chain elongation proceeds normally until, by chance, DNA polymerase inserts a dideoxy nucleotide (shown as colored letters) instead of the normal deoxynucleotide (shown as vertical lines). If the ratio of normal nucleotide to the dideoxy versions is high enough, some DNA strands will succeed in adding several hundred nucleotides before insertion of the dideoxy version halts the process.

Automated DNA sequencing method
At the end of the incubation period, the fragments are separated by length from longest to shortest-using polyacrylamide gel electrophoresis. The resolution is so good that a difference of one nucleotide is enough to separate that strand from the next shorter and next longer strand. A laser constantly scans the bottom of the gel, detecting bands as they move down the gel. Each of the four dideoxynucleotides fluoresces a different color when illuminated by a laser beam. An automatic scanner provides a printout of the sequence. Where the manual method uses radioactive labeling, automated sequencing uses fluorescent tags on the ddNTPs (a different dye for each nucleotide). This makes it possible for all four reactions (dGTP, dATP, dCTP, and dTTP) to be run in one lane, so you can have huge numbers of reactions on one gel. This is a very efficient method.

Automated sequencing gel

The sequenced strand can be read 5' to 3' by reading top to bottom the bases complementary to the those on the gel.

Chromatogram

Each band of fluorescence is read automatically and recorded (see figure ). The DNA sequence is also printed out automatically based on the sequence of colors of the fluorescent peaks that are detected.

Why do we need to sequence DNA?
For the identification of nucleotide sequences of the cloned DNA. For future manipulation of the cloned DNA Identification of restriction enzyme sites within the DNA to provide precise restriction maps. To confirm any changes that has been made by in vitro mutagenesis.

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