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)
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.
DNA Melting Curves
EOC Problem 10: the Hyperchromic Shift in DNA…seeing the transition from ds to ss DNA.
DNA Polymerase: Primern + dNTP => Primern+1 + PP + H+
DNA pol Reaction Needs:
Template, Primer, dNTP’s
ssDNA
Classical SangerDideoxy-DNA sequencing
Reaction:
template, primer
dNTP’s at mM conc.
ddNTP’s at μM conc.
Each reaction in a separate tube
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.
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 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
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.
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.