1. CHAPTER 7 DNA SEQUENCING - INTRODUCTION - SANGER DIDEOXY METHOD - AUTOMATED SEQUENCING - NEXT GENERATION OF SEQUENCING METHODS MISS NUR SHALENA SOFIAN

2. INTRODUCTION 1977: Frederick Sanger along with Allan Maxam and Walter Gilbert developed 2 methods in determining the exact base sequence of a cloned piece of DNA They have made it possible to sequence all 3 billion base pairs of the human genome

3. The Sanger Chain-Termination Sequencing Method Utilization of ddNTPs, primers, single strand of DNA template, DNA polymerase and dNTPs ddNTPs are essentially the same as nucleotides except they contain a hydrogen group on the 3’ carbon instead of a hydroxyl group (OH)

5. REMEMBER: ddNTPs do not permit any DNA synthesis so their inclusion should be just enough with excess of normal dNTPs. Some strands will terminate early; other later A sample containing copies of ssDNA is mixed with primers – bind to primer-annealing site Either the primers or the dNTPs are radiolabeled with 32 P dNTPs and DNA pol. are then incubated allowing DNA pol. to stretch out DNA strand 4 tubes contain different ddNTPs: ddATP, ddGTP, ddCTP, ddTTP

6. The result is a series of fragments of different lengths in each tube All reaction mixtures are electrophoresed on acrylamide gel under denaturing condition – DNA becomes ss Shorter strands move to the bottom of gel more quickly than the longer strands DNA is autoradiographed for visualization – appear as horizontal bands on X-ray film

7. As the DNA is synthesized, dNTPs are added on to the growing chain by the DNA polymerase. However, on occasion a ddNTP is incorporated into the chain in place of a normal nucleotide, which results in a chain-terminating event. For example if we looked at only the "G" tube, we might find a mixture of the following products:

8. DNA Sequencing Example 1

9. HOW TO READ THE SEQUENCE? Read base sequence from bottom to top Known as sequencing ladder

10. DNA Sequencing Example 2 We’ll look at the original method, which used radiolabeled DNA. Small amounts of the dideoxy nucleotides are spiked into a DNA polymerase reaction.

11. There are two tricks to remember: 1. There is more than one DNA strand in the reaction. 2. Not all of the nucleotides are of the dideoxy variety. So multiple terminated products are generated (see red arrows)

12. Then we resolve the population of fragments on a gel. Since we know which dideoxy was used in each reaction, we can read the gel.

13. "A" tube: all four dNTP's, ddATP and DNA polymerase "C" tube: all four dNTP's, ddCTP and DNA polymerase "T" tube: all four dNTP's, ddTTP and DNA polymerase "G" tube: all four dNTP's, ddGTP and DNA polymerase DNA Sequencing Example 3

14. AUTOMATED SEQUENCING Innovation of dideoxy sequencing Instead of having 4 separate tubes, this method only involves one tube containing all four ddNTPs Each ddNTP has different-colored flourescent label attachment Incubate the target DNA with dNTPs and DNA pol. along with ddNTPs and the sample is then loaded into single lane of gel The resulting bands are detected via laser and florescence detector – record amount of fluorescent emission

19. Maxam-Gilbert Sequencing Method Starting material is end-labeled DNA template; later cleave it with base-specific reagents e.g. DMS DNA fragment to be labeled can be 5’ or 3’-end e.g. 32 P One or two of the four bases (either G, A+G, C, C+T) is methylated under mild condition using: - dimethyl sulfate (DMS) for purines - hydrazine for pyrimidines Piperidine is added next: - causing loss of the methylated base - breaking DNA backbone (apurinic site / apyrimidinic site) Electrophorese the products and detect by autoradiography

20. However, with the improvement of the chain- termination method, Maxam-Gilbert sequencing has fallen out of favour due to its technical complexity prohibiting its use in standard molecular biology kits, extensive use of hazardous chemicals, and difficulties with scale-up.

22. HOW THE HUMAN GENOME WAS SEQUENCED??

23. Next Generation of Sequencing Methods 1.Polony sequencing Developed at Harvard, was among the first next- generation sequencing systems used to sequence a full genome in 2005 Combination of an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation- based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1/10 that of Sanger sequencing The technology was licensed to Agencourt Biosciences, subsequently spun out into Agencourt Personal Genomics, and ultimately incorporated into the Applied Biosystems SOLiD platform.

24. 2. Pyrosequencing Developed by 454 Life Sciences454 Life Sciences Amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony The sequencing machine contains many picolitre- volume wells each containing a single bead and sequencing enzymes. Utilizes luciferase to generate light for detection of the individual nucleotides added to the growing DNA, and the combined data are used to generate sequence read-outs This technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other

26. 3. Illumina Sequencing Based on reversible dye-terminators DNA molecules attached to primers on a slide and amplified so that local clonal colonies are formed (bridge amplification)bridge amplification Four types of ddNTPs are added, and non- incorporated nucleotides are washed away Unlike pyrosequencing, the DNA can only be extended one nucleotide at a time A camera takes images of the fluorescently labeled nucleotides then the dye along with the terminal 3' blocker is chemically removed from the DNA, allowing a next cyclefluorescently labeled

27. 4. DNA Nanoball Sequencing To determine the entire genomic sequence of an organismgenomic sequence The company Complete Genomics uses this technology to sequence samplesComplete Genomics Utilization of rolling circle replication to amplify small fragments of genomic DNA into DNA nanoballsrolling circle replication Unchained sequencing by ligation is then used to determine the nucleotide sequence Allowing large numbers of DNA nanoballs to be sequenced per run and at low reagent costsreagent But only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult. This technology has been used for multiple genome sequencing projects and is scheduled to be used for many morereference genome

28. D. Imaging DNA Nanoball Sequencing