L9- DNA structure + analysis

Who coined the term nuclein

• Miescher

• discovered nucleic acid (contains C,H,O,N,P) found in nucleus of all cells→ Nuclein

Griffiths and Avery expts

• To determine whether protein/DNA contains genetic material

• Griffiths transformation expt (mice die S+R, heat killed S+R)

  • R→S

• Avery’s expt→ DNA=transforming component

  • bc DNA from dead S destroyed w/ DNase→ no transformation

Bacteriophage T2

• phages are 50% protein and 50% DNA

• Hershey and Chase

  • Phages labelled w/ 32P-DNA 35S- protein

  • E.coli infected w/ phages→ radioactivity recovered in host and passed on to phage progeny

    • 32P in pellet

  • With 35S→ radioactivity in phage ghosts + NOT passed on to progeny

• ³²P radioactivity was found in the pellet, meaning the labeled DNA entered the bacterial cells.

• ³⁵S radioactivity stayed mostly in the supernatant, meaning the protein coats remained outside.

How does viral genetic material vary

• Depending on virus

• Some use DNA/RNA

• cricular/linear chroms

• small/large segments

• single/ds

Nucleoside vs nucleotide

• nucleoside= sugar + n base NO phosphate

• nucleotide= sugar (pentose), N base, P group

Polynucleotide chains have

• polarity

Phosphodiester linkages bw

• 3’ OH and 5’ Phosphate group

• (synthesis occurs 5’-3’ but adds on to 3’ end)

Chargaff

• 50% base= purines, 50% bases= pyrimidines

• A=T

• C=G

X ray diffraction studies

• Franklin + Wilkins

  • regularities of 0.34 nm and 3.4 nm

  • DNA is a helical structure

Watson and Crick model of DNA

• 2 polynucleotide chains wound around each other in a right handed helix

• 2 chains antiparallel

• Sugar-P backbones on outside of double helix

• Bases connected by H bonds

• Bases 0.34 nm apart, 10 bases= 1 full turn

• Grooves of unequal size form bw backbones

• double helix diameter= 2 nm

RNA structure

• single stranded

  • will bind to itself when possible (folds on itself)

• Many diff forms of RNA in cell

  • rRNA, tRNA, mRNA

    • miRNA, snoRNA, etc

Different DNA structures

• B-DNA→ watson and crick model right handed helix

• A-DNA→ dehydrated B DNA adopts this conformation (dry envt) right handed helix

• Z-DNA→ left handed helix, artifical maybe in our genome

DNA applications

• Can store hrs of video or pictures

• Origami (make shapes)

Melting Temp (Tm)

• DNA can be denatured by heat/pH stress

  • Tm

  • Diff UV absorbency single vs ds (ds bases hidden so absorb less)

  • Hyperchromatic shift (change in absorbance (increase) as ds turns into ss)

• Temp where absorbance is in middle of ds and ss DNA

Factors affecting Tm

• GC content (3 H bonds more stable harder to break than 2 AT)→higher Tm

• Base stacking ( smaller role but within a strand GCs next to each other more stable, increasing Tm)

• Degree of complementarity (how well strands stuck to each other)

Hybridization (shorter vs longer seqs and annealing)

• After denaturation DNA strands can reanneal

  • shorter anneals faster than longer

  • Highly complementary (repetitive) seqs anneal faster than diverse ones

What is FISH used for

• identifying chromosomal location of a DNA sequence

• ex) Downs syndrome bc will see 3 signals instead of 2

What’s electrophoresis used for

• Separating DNA and RNA fragments by size

• Smaller fragments migrate through gel faster than larger fragments