BENG 183 Trey Ideker (the details )

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1 BENG 183 Trey Ideker (the details )

2 (1) Devils in the details: Sequencing topics to be covered in today s lecture DNA preparation prior to sequencing Amplification: vectors or cycle sequencing PAGE and the 1000 bp read limit Polymerases used for sequencing Capillary sequencing DNA synthesis What reagents go into a sequencing reaction and what is the purpose of each?

3 A bit of history: DNA sequencing in 1977 Maxam/Gilbert (Chemical) Enzymatic (Sanger) First complete genome sequence of phage ΦX174 by Sanger The standard method today is the Sanger dideoxy method Nobel prize in 1980

4 DNA preparation Many steps are required before DNA samples are loaded on gels: DNA isolation Fragmentation Amplification (bacterial vectors, PCR, cycle sequencing) Re-isolation of DNA (if vector amplified) Most of these have also been automated

5 Vectors for sequencing Sequencing requires a single stranded template DNA to be sequenced is in a vector such as M13 or puc Most vectors have universal priming sites flanking one or more restriction enzyme sites Propagation in bacteria leads to exponential amplification of DNA

6 M13 vectors for DNA sequencing Circular DNA modified from the genome of the M13 bacteriophage. Transitions through both single and double stranded forms, making it ideal for sequencing applications Double stranded DNA is the replicative form and is used for cloning. DNA packaged in phage capsid is single stranded.

7 M13 vectors for DNA sequencing From Recombinant DNA 2 nd edition, Watson et al.

8 Cycle sequencing PCR-like approach; Vector/bacteria free Accommodates a double stranded DNA template Requires sequence-specific primers 25 cycles of {denaturation, annealing, extension} Provides linear (not exponential) amplification of the DNA template Run a Shockwave animation of cycle sequencing

9 Polyacrylamide gels Gel is 7M urea and 4-8% acrylamide 1600 volts, heats gel to 65 C ~60 cm long Denaturing gel (two reasons why?) Resolves single DNA bp differences up to 1000 bp in length (why not longer???)

10 Polymerase Enzymes for DNA sequencing Processivity: average # of nucleotides synthesized before enzyme dissociates

11 Sequencing Dyes What are the desired spectral characteristics of fluorescent dyes used for sequencing? Common excitation wavelength Distinct emission wavelenths

12 Automated sequencing Detection of DNA band is automated Uses enzymatic (Sanger) method Uses fluorescent labels instead of radioactivity Label is excited by a laser at bottom of gel The detector scans horizontally across the base of the gel so as to scan several sequencing lanes Labels are either on the primer or attached to the ddntp terminators

13 Automating the sequencing process Scanning Detector (Photo multiplier tube PMT) LASER

14 Fluorcnt. gel scanned image

15 Trace of scanned image

16 ABI 371 and 3700 sequencers

17 Problems with gel-based sequencing Preparation of gels is labor intensive DNA sequencing speed is related to the strength of the applied electric field High voltage causes Joule heating of the gel and utter meltdown CAPILLARY SEQUENCING partially solves these problems

18 Sequencing by capillary electrophoresis

19 Megabase capillary sequencer

20 The brown wire-like loops are the 96 individual capillary tubes. They are made of glass and coated with brown plastic. The samples are loaded from below this level on the left side and the DNA is electrophoresed towards the right. The matrix is injected into the tubes after each run and it travels from right to left when filling the tubes.

21 DNA Synthesis Proceeds in the 3 to 5 direction Starts with a column with the 3 base attached The reactive hydroxyl OH group is protected by a dimethoxytrityl (DMT) group Each cycle proceeds as follows: Detritylation: The DMT group is removed to generate a free 5 hydroxyl Coupling: The next base is added together with a coupling reagent Capping: Any remaining unreacted (uncoupled) bases are capped to block further reaction Oxidation: Coupling produces a trivalent phosphite bond which must be oxidized to phosphate using a sulphurizing agent

22 Oligo synthesis: Detritylation

23 Oligo synthesis: Coupling

24 Oligo synthesis: Capping & Oxidation

25 DNA synthesis continued To finish, the oligo is: Cleaved from the column Incubated in ammonium hydroxide to remove all protecting groups Ethanol precipitated Quantitated in a spectrophotometer at OD 260 Optionally gel purified Two common instruments are: ABI 3900 DNA synthesizer (48 oligos per run in the range of bases long) ABI 394 DNA synthesizer (4 oligos per run up to 160 bases long) The whole process typically takes hours

26 An example instrument

27 Common applications requiring synthesized DNA oligonucleotides DNA sequencing (cycle seq, not vectors) Polymerase Chain Reaction (PCR) Site-directed mutagenesis Cloning small regions of a gene, e.g. transcription factor binding sites, epitope tags, membrane domains Microarray fabrication!!!

28 Oligonucleotide Primer Design bases long for sequencing, sometimes longer for PCR At least 40 bp outside of the region of interest Melting temperature T m ~ 50 C. Rule of thumb: GC content of ~50% T m =2(A+T) + 4(G+C) No long runs of a single base No dimer or hairpin loop formation Computer programs are available for primer design, e.g. Primer3, Primer Designer, etc.

29 Some common oligonucleotide primers PRIMERS M13Forward M13F(-40) M13Reverse M13R(-48) T7 promoter T7 terminator T3 promoter SP6 promoter BGH Reverse SEQUENCE 5'-GTAAAACGACGGCCAGT-3' 5'-GTTTTCCCAGTCACGAC-3' 5'-CAGGAAACAGCTATGAC-3' 5'-AGCGGATAACAATTTCACACAGGA-3' 5'-TAATACGACTCACTATAGGG-3' 5'-GCTAGTTATTGCTCAGCGG-3' 5'-ATTAACCCTCACTAAAGGGA-3' 5'-GATTTAGGTGACACTATAG-3' 5'-TAGAAGGCACAGTCGAGG-3'