Nucleotides and Nucleic Acids Part 2 (and a little bit from Chapter 9) Chapter 8

Nucleotides and Nucleic Acids, Part 2 Learning Goals 1.Single, double, triple, quadruple stands. 2.Denaturation and renaturation (annealing) of DNA 3.Chemical changes to DNA = mutations and others. 4.Sanger DNA sequencing. 5.Next Gen Sequencing (in Chapter 9) 6.Restriction enzyme specificity and activity (in Chapter 9)

EOC Problem 5: all about hairpin structures and differences.

Prokaryote and Eukaryote Gene Structure

single stranded RNA Phosphate: oxygen atoms are green phosphorus atoms orange Ntide-bases yellow

RNA helix different from DNA helix: 1. RNA helix in A form (B form not seen yet) 2. G can pair with both C and U, A pairs with U 3. 2’ OH can H-bond with other stem+loops.

E. coli RNase P, RNA component Ribozyme

t-RNA

Figs from Chapter 27

E. coli 16S rRNA

Melting and Annealing

DNA Melting Curves EOC Problem 10: the Hyperchromic Shift in DNA…seeing the transition from ds to ss DNA.

DNA Melting in the TEM

Chemical Change of DNA happens

EOC Problem 8: change in dsDNA after depurination.

UV  Thymine Dimers

These are all Mutagens that Change DNA

DNA Polymerase: Primer n + dNTP => Primer n+1 + PP + H + DNA pol Reaction Needs: Template, Primer, dNTP’s ssDNA

If this is incorporated, it brings about strand termination Dideoxy-NTP lack 3’-OH

Classical Sanger Dideoxy-DNA sequencing Reaction: template, primer dNTP’s at mM conc. ddNTP’s at μM conc. Each reaction in a separate tube

DNA Sequencing - Electrophoresis

EOC Problem 13 Is all about Sanger Sequencing Logic and EOC Problem 14 is applied to a sequencing gel. These are the basic logic used and applied in different ways to Next Gen Sequencing…which is part the very little bit we are adding in from Chapter 9 in 6 th Ed only.

Dideoxysequencing with Fluorescent ddNTP’s

Sequencing Products

454 or Pyro-Sequencing Two types of Beads, in each well: one with DNA (the big yellow one), many small ones with the enzymes (little reddish ones) SO 4 = luciferin

Pyrosequencing

Pyrosequencing Pico-Titer Plates 43 μm Loading the Enzyme Beads

Pyrosequencing Overview Sample Genomic DNA + BACs Nebulized Fragments Adapters Added Water in Oil Microreactors One bead/well Picotiter Plate Pyrosequencing: sequential addition of dNTPs one at a time. Imaging measures the light per well with each successive different dNTP addition. We didn’t go over this

Pyrosequencing Data Output Diagram from One Well Bases

Pyrosequencing from Chapter 9 pgs

Look At Light from One Well with Each N-tide Added There is another system using colored (fluorescent) nucleotides that uses blocking agents on the N-tides not incorporated. Illumina system, basically the same, just another variation.

Ion Torrent Sequencing

Ion Torrent Read … from one Well

Synthetic DNA

Nucleotides

ATP

Coenzyme A

NAD +

FAD

Signaling Nucleotides

Nucleotide Intracellular Concentrations * NucleotideConc, μM Nucleotide Conc, μM ATP3,000GTP923 ADP 250GDP128 AMP 105GMP 20 dATP 175dGTP122 dTTP 77dCTP 65 UTP 894CTP515 cAMP 6cGMP nd ppGpp 31 NAD + 790NADP 54 NADH 16NADPH146 FAD 51FMN 88 AcCoA 231SuccCoA 15 in Salmonella enterica subsp Typhimumrium from Bochner and Ames, 1982, J. Biol. Chem 257:

1.dATP, dTTP, dCTP, dGTP, ddTTP 2.dATP, dTTP, dCTP, dGTP, ddGTP 3.dATP, dCTP, dGTP, ddTTP 4.dATP, dTTP, dCTP, dGTP Problem 14 – get the results

Things to Know and Do Before Class 1.Sequence patterns, ssRNA, dsRNA…and more. 2.RNAs: Ribozymes, t-RNA, r-RNA. 3.Temperature denaturation and annealing. 4.Mutagens. 5.Nucleotides and coenzymes, signaling nucleotides. 6.DNA Sequence determination: a.Classical and modern Sanger Sequencing b.Pyro-sequencing. c.Ion Torrent (pH) sequencing. 7.EOC Problems to do: 5, 8,10,12-15.