DNA

1. Chemical structure 2. Physical structure 3. Replication

Copyright (c) by W. H. Freeman and Company

Basic Techniques -DNA extraction -Detection -Cloning -Hybridization (microarrays) -Sequencing

All nucleotides have a common structure

dNTP

Five principal bases in nucleic acids

A, G, T, C are present in DNA A, G, U, C are present in RNA

What are the 2 chemical difference between DNA and RNA?

NTP, dNTPs and ddNTPs

Copyright (c) by W. H. Freeman and Company

Native DNA is a double helix of complementary antiparallel chains held together by:

What is the complimentary sequence to:

Sequence: 5-ATTGGC-3

10.5 bp/turn

11 bp/turn

12 bp/turn

Ethidium Bromide (intercalating agent) - fluorescent dye that binds DNA

DNA can undergo reversible strand separation

Hybridization

Force that maintain DNA as a double strand.

Denaturing agents displace H-bonds and separate two strands of double helix. -formamide -high pH (NaOH) -high temperature

Analysis of DNA denaturation

Tm= temperature at which 1/2 the bases dsDNA sample have denatured

Many DNA molecules are circular and local unwinding of circular DNA can produce supercoiling

supercoiled

relaxed

Physical Structuree

Chromosome structure Genomes

Copyright (c) by W. H. Freeman and Company

The genome of Rhizobium leguminosarum

Genome Biology 2006, 7:R34

DNA Replication

Copyright (c) by W. H. Freeman and Company

Semiconservative replication

Copyright (c) by W. H. Freeman and Company

DNA

DNA Synthesis
H H

Incoming dNTP

PPi 2P

Q: If DNA can only be synthesized in a 5 to 3 direction, and both strands are simultaneously replicated, how can this occur? 5 3
growing fork 5

-Problem-

3 5

5 3

The Solution

(Okazaki fragments)

DNA Replication Requirements

How does DNA replication differ from a PCR reaction? DNA Replication
1. 2. 3. 4. DNA Polymerase DNA Template primer of DNA or RNA dNTPs

PCR reaction
1. 2. 3. 4. 5. DNA Polymerase DNA Template 2 primers of DNA dNTPs buffer

How PCR Works

Reagents (set-up)

PCR is an artificial way of doing DNA replication

Template DNA 2 Primers (flank the DNA fragment to be amplified) dNTPs Taq DNA Polymerase (thermally stable DNA polymerase) Buffer

Machine (changes temperatures): Melt DNA (denaturing) Hybridize Primer (annealing) Polymerization (extension)

Summary
Chemically important regions of nucleic acid for molecular diagnostic techniques - structural differences between DNA and RNA - overall charge (separation; gel electrophoresis) - NTP, dNTP (for PCR), ddNTP (for sequencing) - Melting temperature -Tm (for hybridizations, PCR) - Proper way to write a sequence (for sharing sequence data, probes) - packaging of DNA (replication) - plasmids vs. chromosomes (linear vs. cirucular) DNA Replication: in vivo vs. in vitro (PCR) - needed reagents