1. Working with DNA Lecture 8 1

2. Structural Predictions – Chou Fasman 2 Guidelines 1.A cluster of 4 helix forming residues (H  or h  out of 5 sequential residues will nucleate a helix. i.Once the average value of 4 sequential residues falls below 1, the helix is broken. 2.A cluster of 3 sheet forming residues (H  or h  out of 5 sequential residues will nucleate a sheet. i.Once the average value of 4 sequential residues falls below 1, the helix is broken. 3.If both helix and sheet are predicted, the highest average value will be preferred. http://www.biogem.org/tool/chou-fasman/

3. 3 AAHelixSheet Gln1.11h1.10h Leu1.21H1.30h Met1.45H1.05h Thr0.83i1.19h Trp1.08h1.37h Ala1.42H0.83i Ser0.77i0.75b Thr0.83i1.19h Pro0.57B0.55B Cys0.70i1.19h Gln Leu Met Thr Trp Ala Ser Thr Pro Cys Structural Predictions – Chou Fasman

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5. 5 Peptide Synthesis Fmoc Activated Ester

6. 6 Peptide Synthesis

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8. Working with DNA 8 What we need for PCR Reaction: + Primers and DNA Polymerase Polymerase Chain Reaction (PCR) revolutionized the fields of biology and biochemistry

9. PCR – the basic reaction 9

10. Molecular Cloning Strategies 10 Biochemists commonly use E. coli to express proteins Gene must be introduced in a way that E. coli will replicate the DNA and transfer it to daughter cells https://www.neb.com/tools-and-resources Requirements for an expression plasmid Antibiotic resistance RNA polymerase binding site Ribosome Binding Site Origin of Replication

11. Primers – the most important part of experiment design 11 The exponentially amplified sequence falls between the primers Aim for T m values around 60 °C. The two primers should have roughly the same T m Directionality is key! You can add sequences on the 5’ or 3’ end using primers. This is very common for cloning (adding restriction sites)

12. DNA Sequencing 12 What we need for PCR Reaction: + Primers and DNA Polymerase Polymerase Chain Reaction (PCR) revolutionized the fields of biology and biochemistry

13. PCR Termination 13 2’,3’-dideoxyATP

14. DNA Sequencing 14

15. DNA Sequencing 15

16. DNA Sequencing 16

17. Electropherograms 17

18. Next Generation Sequencing 18 Recent advances in sequencing methods has made genome sequencing very fast and cheap! Human Genome Project (used chain termination method) took 10 years to complete and over $300 million! Next-Gen techniques allowed the genome of James Watson (of Watson and Crick fame) – the 3 rd sequenced human genome) to be sequenced in 2 months for less than $1M.

19. Next Generation Sequencing - Pyrosequencing 19 DNA is randomly sheared by sonication 300-500 bp fragments produced dsDNA is melted apart to form single strands. ssDNA are bound to DNA “capture” Beads SPRI Beads = solid phase reversible immobilization This capture reaction happens under REALLY dilute conditions (< 1 DNA molecule per bead) Under high salt conditions (2.5 M NaCl) and PEG, DNA binds to the carboxylate. Believe it, it happens. DNA is amplified on the bead by PCR until ~10 million copies are present

20. Next Generation Sequencing - Pyrosequencing 20 Beads are transferred to a fiber optic slide with one bead per well. Each well is 75 picoliters and one slide contains 1.6 million wells Pyrosequencing reactions occur in these wells. 1.One nucleotide (let’s say dGTP) is added to the slide. 2.A ‘burst’ of light is seen (sensed through the fiber optic cable) if the dNTP is complementary. 3.Wash off residual dGTP 4.Add dATP – burst – wash 5.Add dCTP – burst – wash 6.Add dTTP – burst – wash 7.Back to 1 st step

21. Next Generation Sequencing - Pyrosequencing 21 Preparation for next step Once sequencing reactions are done, genome sequence is pieced back together (shotgun approach)