4 DNA : it’s structure, replication, and related assays

The components of a nucleotide Nitrogen containing base, including A, T, G, and C in DNA A, U, G, and C in RNA Ribose (5 carbon atoms) deoxyribose in DNA ribose in RNA Connect neucleotides

Ribose 1’ has base: A, T, G, C (in DNA) A, U, G, C (in RNA) 2’ is H in DNA, OH in RNA 5’ has phosphate group to connect with 3’ of another nucleotide Deoxyribose

Why DNA , instead of RNA, carries the genetic information The 2’ OH in the ribose attacts the phosphodiester bond between nucleotide molecules and results the hydrolysis of RNA. DNA has no OH on 2’ and is more stable.

Nucleotides are joined by linking the phosphate on the 5’ end of the deoxyribose with the 3’ end of next deoxyribose. Imaging it as the floors of a department store.

Makes DNA negatively charged

Native DNA is a double helix of complementary antiparallel strands. A base pair Native DNA is a double helix of complementary antiparallel strands. Anti-parallel: one strands goes from 3’ to 5’ and the other goes from 5’ to 3’

G C

Deoxyribose backbone Deoxyribose backbone

Heating separates DNA into single strands DNA melts or denatures (meaning separation of double strands) when temperature increases.

GC contents affect the DNA melting temperature (Tm) As GC contents increases, Tm increases.

Annealing (hybidizing) of single stranded-DNA Annealing: the rejoining of separated single strands of DNA to form double helix Hybride DNA: artificial doubled stranded DNA formed by 2 single stranded DNA from different sources

DNA techniques To purify genomic DNA to separate dna molecules by it’s size to detect DNA molecules by labeling

Obtain genomic DNA 1 Lysis of cell Use chemical (detergents..) or physical (ultrasonic..) methods 2 Centrifugation To remove cell debris (large molecules) 3 Extraction To remove proteins and other hydrophobic molecules 4 Remove RNA Use ribonuclease to degrade single strand RNA

Obtain genomic DNA 3 extraction 4 remove RNA

Obtain genomic DNA The interaction between DNA (negatively charged) and glass or other positively charged material is widely used to separate DNA from solution.

DNA purification kit (most convenient )

Measure the concentration of your DNA DNA concentration (µg/ml) = (OD 260) x (dilution factor) x (50 µg DNA/ml)/(1 OD260 unit) RNA concentration (µg/ml) = (OD 260) x (dilution factor) x (40 µg RNA/ml)/(1 OD260 unit)

Electrophoresis knows the size of your DNA? DNA carries negative charge on their phosphate group, therefore, it moves toward the positive electrode. Q: can we use free electrophoresis to separate DNA by its size?

Size does matter - - - - - - - - - - - - Small DNA, less charge - Mobility increases with charge number but decreases with molecule size - - - - - - - Longer DNA, more charge Q: can we use free electrophoresis to separate DNA by its size? A: no, the effects of charge and size cancel each other

Gel Electrophoresis knows the size of your DNA

Gel Electrophoresis knows the size of your DNA The DNA fragments are stained with ethidium bromide (EtBr)

Detection by radioactivity

Duplication of DNA prior to cell division DNA replication Duplication of DNA prior to cell division

Torsional stress in DNA can be relieved by enzymes Many prokayrotic genomic DNAs and viral DNAs are circular molecules. Circular DNAs also are present in mitochondria and chloroplast in eukaryotic cells. We need to relax the DNA in order to carry out the replication. DNA Gyrase

Enzymes needed to OPEN DNA strands 29 DNA helicase Requires more enzymes than gyrase and helicase

DNA helicase and strand binding protein SSB prevents the annealing of complementary strands

Replication overview III

DNA polymerase in action III Primer: a short piece of DNA sequence that facilitates the initiation of polymerization Primer is made by primase.

Synthesis goes from 5’ to 3’ Both DNA and RNA synthesis proceed from 5’ to 3’ due to the molecular structure of nucleotide monomer.

Replication fork : the new strand of DNA that is synthesized continuously III : fabricate primer : the new strand of DNA that is synthesized in short pieces and then joined together later

Replication fork in action

Okazaki fragments: short pieces of DNA that make up the lagging strand DNA polymerase I: enzyme that makes small stretches of DNA to fill gaps between Okazaki fragments DNA ligase: an enzyme that joins DNA fragments end to end I

Semi-conservative replication

Semi-conservative replication: evidence

Division of circular bacteria chromosomes

Division of bacteria cells

Division of higher organism cells Eukaryotic cells have chromosomes with complicated structure. The replication of eukaryotic chromosomes initiate at multiple origins spaced along chromosomal DNA.

Polymerase Chain Reaction (PCR) To amplify a specific region (usually known) of DNA sequence from a complex mixture. PCR depends on the ability of reversible separation (melting) of double –stranded DNA.

Polymerase Chain Reaction (PCR) The copy number of targeted DNA can be calculated by 2x x is the cycle number. In order to get accurate amplified sequence, the selection of primer is important. Taq polymerase: most common polymerase used in PCR dNTP: deoxynucleotide

Why we use hydrophobic surface when combing DNA?

Why we use hydrophobic surface when combing DNA? DNA adsorbs onto this hydrophobic surface by one extremity in a “mushroom” state. This means that the adsorbed DNA has only one attachment point with the surface and retains its fluctuating coil conformation. The reason the DNA binds to the hydrophobic surface is still a matter of discussion. One explanation relies on pH-induced denaturation of the DNA ends, which then expose the hydrophobic part of bases and thus strongly interact with the surface (27) Allemand J F, Bensimon D, Jullien L, Bensimon A, Croquette V (1997) Biophys J 73:2064–2070