1. First generation Gene Sequencing
Jessie Marlenee
BIOL 546

2. Recap Watson & Crick discover DNA double helix 1953
Restriction Enzymes
Southern Blotting
Cloning

3. Initial sequencing efforts
Proteins
RNA
Holley and colleagues (1965)
alanine tRNA from Saccharomyces cerevisiae
Wu and Kaiser ( )
Incorporation of radiolabeled nucleotides into strand to determine sequence
Walter Fiers' laboratory ( )
Coat protein of bacteriophage MS2, followed by complete genome four years later (3569 nucleotides of ssRNA)
Replacement of 2D fractionation
DNA discovered in 1953 but did not have ability to read “sequence” order of DNA.4
Issues with DNA that delayed the ability to sequence
-Chemical properties of DNA molecules so similar so difficult to separate
-Chain lengths of naturally occurring DNA much longer than proteins which had been previously sequenced
-No base specific DNAases known
Thus, initial sequencing efforts focused on RNA because
-molecules small and individual types could be purified
-RNAases with base specificity were already known so methods similar to those used for proteins could be used
1965- Holley & Colleagues
-Sequenced first nucleic acid molecule
-tRNA from Saccharomyces cerevisiae
Sanger and colleagues developed technique based on detection of radiolabeled partial-digestion fragments using two-dimensional fractionation
Walter Fier’s and colleagues
-Used method to produce first complete protein-coding gene sequence in 1972
Coat protein of bacteriophage MS2
Followed by the complete genome four years later (1976)
Purification of bacteriophages with DNA genomes continued…
Wu & Kaiser
-used DNA polymerase to fill overhanging “ends” on phage lamda DNA with radioactive nucleotides, supplying 1 at a time and measured incorporation to determine the sequence of DNA
-Partial sequence in ‘68 followed with complete 12 base sequence in 1971
2D Fractionation (multi step- electrophoresis and chromatography) replaced with single separation by polynucleotide length via electrophoresis through polyacrylamide gels
-greater resolving power
-lead to the first sequencing techniques
Kaiser, A. D. and Wu, R

4. Sanger’s “Plus and Minus” technique (1975)
Fred Sanger and Alan Coulson’s “plus and minus” technique (1975).
Used DNA polymerase to synthesize/build DNA from a primer, while including radiolabeled nucleotides before doing 2 reactions. Both reactions terminated in a sequence specific manner.
“plus” reaction- single type of nucleotide present so all extensions end with that base
“minus” reaction- 3 types of nucleotides (remaining 3) terminates at position before next missing nucleotide
8 aliquots, ran on polyacrylamide gel. Compare 8 lanes.
Used technique to sequence first genome: Bacteriophage Φx174 “phi x”.
Still used in labs today as a positive control genome.
Problem with this method is difficulty in determining length of homopolymer runs

5. Maxam and Gilbert’s Chemical cleavage technique (1977)
Maxam and Gilbert developed “chemical cleavage” technique in 1977
-Did not rely on DNA polymerase to generate fragments
-Double stranded DNA restriction fragment radiolabeled on one end with 32P
-DNA treated with chemicals that cleave it at specific bases (4 reactions)
Cleaves both purines (A+G)
Cleaves at Guanines (G)
Cleaves at pyrimdines (C+T)
Cleaves at Cytosines (C)
-Gel electrophoresis produces bands for every sequence position (including homopolymer runs)
-2 problems with method
Use of hazardous chemicals (hydrazine is neurotoxin)
Lots of DNA needed and 4-8 reactions needed
Matthews and van Holde: Biochemistry, 2nd edition

6. Sanger’s “chain termination” technique (1977)
Also known as the dideoxynucleotide method or sanger sequencing
Technique developed by Sanger and Coulson in December 1977
-Method involved using chemical analogues of deoxyribonucleotides (dNTPs) (monomers of DNA strands)
-ddNTPs lack the 3’ hydroxyl group necessary for extension of DNA, cannot match with 5’ phosphate of the next dNTP…thus terminating the chain (chain termination” method
-Four simultaneous reactions, one with individual ddNTP base
-Mixing ddNTPs labeled with 32P into a DNA extension reaction at a lower concentration (~1%) of dNTPs produces DNA strands of each possible length
-Products ran on polyacrylamide gel in four lanes then autoradiographed, sequence can be read from bottom of gel to top (5’ to 3’)
-Less toxic than Maxam-Gilbert technique, more efficient. Became most widely used method of sequencing
Improvements made to method
-Switch from labeling DNA with 32P (radioactive isotope) to fluorescent dyes

7. Video showing Sanger Sequencing

8. 1980 Nobel prize in chemistry
½ Awarded to Paul Berg
¼ Awarded to Walter Gilbert
¼ Awarded to Frederick Sanger

9. References
Franca, L. T., Carrilho, E., and Kist, T. B A review of DNA sequencing techniques. Q. Rev. Biophys. 35:169–200.
Guzvic, M The history of DNA sequencing. J Med Biochem. 32:301–12.
Heather, J. M., and Chain, B The sequence of sequencers: The history of sequencing DNA, Genomics. 107:1-8.
Hutchison C. A. III DNA sequencing: bench to bedside and beyond. Nucleic Acids Res. 35:6227–6237.
Holley, R. W., Apgar, J., Everett, G. A., Madison, J. T., Marquisee, M., Merrill, S. H., Penswick, J. R. and Zamir, A Structure of a ribonucleic acid. Science. 147:1462–1465.
Kaiser, A. D. and Wu, R Structure and function of DNA cohesive ends. Cold Spring Harb. Symp. Quant. Biol. 33:729–734.
Mardis, E. R Next-generation sequencing platforms. Annu. Rev. Anal. Chem. 6:287–303.
Maxam, A. M. & Gilbert, W A new method for sequencing DNA. Proc. Natn. Acad. Sci. 74:560–564.
Sanger, F. and Coulson, A. R A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J. Mol. Biol., 94:441–448.
Sanger, F., Nicklen, S. & Coulson, A. R DNA sequencing with chain-terminating inhibitors. Proc. Natn. Acad. Sci. 74:5463–5467.
The Nobel Prize in Chemistry NobelPrize.org. Nobel Media AB Thu. 13 Sep
Wu, R. and Kaiser, A. D Structure and base sequence in the cohesive ends of bacteriophage lambda DNA. J. Mol. Biol., 35:523–537.

10. Questions?