DNA Sequencing The DNA from the genome is chopped into bits- whole chromosomes are too large to deal with, so the DNA is broken into manageably-sized overlapping segments. The DNA is amplified by cloning into bacteria (PCR doesn’t produce enough and requires sequence information for the primers). It is then denatured (ie. melted), so that the two strands split apart.

Denatured DNA is added to reaction mix with: a primer (to start complementary pairing), DNA polymerase nucleotides including special ones called dideoxynucleotides. These special nucleotides do not allow further nucleotides to be added to the chain. So in a mix with dideoxy-A, every time a dideoxy-A is added (small proportion of As), the reaction ends. This results in different length fragments. The dideoxynucleotides are fluorescently tagged. Fragments can be separated out on a gel by electrophoresis and their length calculated.

Sequencing DNA involves: Amplifying it by PCR or cloning Chopping it up into manageable bits Replicating it with fluorescently-tagged dideoxynucleotides Running the different length fragments on a gel and reading this Assembling the pieces (sequences of manageable bits). Shotgun sequencing is faster than mapping-based assembly methods.

DNA sequencing – preparation In order to sequence a piece of DNA, first need to amplify it. This is sometimes done by a process called polymerase chain reaction (PCR).

DNA Sequencing DNA sequencing is a common procedure Dideoxy method (Sanger’s Method) Chain termination method Best for small DNA segments Whole genome shotgun sequencing Sequence human genome Fragments larger DNA strand to manageable chunks Pyrosequencing Sequence by synthesis Accurate and fast

DNA Amplification - cloning An alternative to PCR is to insert the piece of DNA into the DNA of a bacterium. Replicating the bacterium thus replicates the DNA. Cf. recombinant DNA technology

Sequencing using gel electrophoresis Here is a gel with 28 DNA samples: green bands represent A, blue C, yellow G and red T. molecules move faster.

Sanger Di-deoxy method Sequencing is used to determine the precise order of nucleotides in a DNA molecule. The Sanger di-deoxy method involves the synthesis of DNA by a DNA polymerase. DNA synthesis is terminated at specific nucleotides by the incorporation of di-deoxy nucleotides that are missing the 3’ OH.

Sequence analysis Four different reactions produce DNA fragments that are terminated (randomly) at each of the four nucleotides. These samples are resolved by electrophoresis. The shortest fragments, those terminated closest to the primer, run faster than the longer fragments.

DNA sequencing reactions can be radioactively labeled. A C G T DNA sequencing reactions can be radioactively labeled. Bands detected by X-ray film exposure. Sequence can be read in the 5’ to 3’ direction from the bottom of the image towards the top. A A T C T A A C G

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Automated DNA sequencers use dideoxy terminators labeled with four different fluorescent dyes. All four reactions can be carried out simultaneously in a single reaction.

Fluorescently tagged fragments are resolved by capillary electrophoresis and detected by a flourometer. The DNA sequence is read automatically.

Beckman CEQ 2000, 8 capillary ABI Prism 3730, 96 capillary

Human Genome Sequencing Human genome project –approximately 3x109 bases (As, Cs, Gs and Ts) in the 23 chromosomes. Extraction of useful information from this list and genome sequence of other organisms relies on computer-intensive data handling – Bioinformatics.

Shotgun sequencing Shotgun sequencing dispenses with the need for mapping and so is much faster. It involves chopping the DNA into fragments of size c. 2000 base pairs (bps) and 10000 bps, sequencing the first and last 500 bps of each fragment. It then uses computer algorithms to assemble the entire sequence from the sequenced fragments.

3200 s. cerevisae= yeast (sequenced 1997); arabidopsis thaliana= plant (2000); c. elegans = nematode worm

Databases of sequence information Internet has become a vital resource in making sequence data generally available to the biological community at large. Examples: GenBank (www.ncbi.nlm.nih.gov/Genbank), EMBL (www.ebi.ac.uk/embl), DDBJ (www.ddbj.nig.ac.jp). Used for: gene prediction, protein structure/ function prediction, homology searching

Conclusions Sequence data is stored in online databases Extracting useful information and patterns from such data is part of bioinformatics and often employs intelligent systems techniques.

2nd Generation: Pyrosequencing Sequencing by synthesis Advantages: Accurate Parallel processing Easily automated Eliminates the need for labeled primers and nucleotides No need for gel electrophoresis