Nucleotides and Nucleic Acids

Part 2(and a little bit from Chapter 9)

Chapter 8

Nucleotides and Nucleic Acids, Part 2Learning 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

t-RNA

Figs from Chapter 27

E. coli16S 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: Primern + dNTP => Primern+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 SangerDideoxy-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 6th 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)

SO4=

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

Bas

es

Pyrosequencing from Chapter 9pgs 339-342

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 Sequencing

Ion Torrent Sequencing

Ion Torrent Read … from one Well

Synthetic DNA

Nucleotides

ATP

Coenzyme A

NAD+

FAD

Signaling Nucleotides

Nucleotide Intracellular Concentrations*

Nucleotide Conc, μM Nucleotide Conc, μM ATP 3,000 GTP 923

ADP 250 GDP 128

AMP 105 GMP 20

dATP 175 dGTP 122

dTTP 77 dCTP 65

UTP 894 CTP 515

cAMP 6 cGMP nd

ppGpp 31

NAD+ 790 NADP 54

NADH 16 NADPH 146

FAD 51 FMN 88

AcCoA 231 SuccCoA 15

in Salmonella enterica subsp Typhimumrium

from Bochner and Ames, 1982, J. Biol. Chem 257:9759-9769

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

2019

17

14

12

98

4

1

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 Sequencingb. Pyro-sequencing.c. Ion Torrent (pH) sequencing.

7. EOC Problems to do: 5, 8,10,12-15.