Lec 14 - Nucleic Acids; DNA Methods Flashcards
nucleotide monomer numbering (1’-5’)
(sugar is 5’C ribose numbered in 5 member ring)
1’ bound to base
2’-OH (rna) or 2’-H (dna)
3’-OH + 5’-P join backbone
- 1’ base; 5’ sticks out
for orientation:
RNA vs DNA
structure due to change in confirmation due from OH group
RNA = C3’- endo A form
- 2’-OH too bulky… pushes into 3’ endo
- puckering happens at 2’ or 3’
DNA = C2’- endo B form
Double-stranded B-form DNA
3 values to memorize + major vs minor groove
- 10.5 bp per turn
- 3.4 A between each bp
- 20 A width
major groove
- 4 bp available (sequence specific binding; sensitive to identity of bp)
minor groove
- similar h-bonding patterns regardless of bp
Monitoring denaturation
sigmoid curve (backwards s)
- reduce in base stacking –> increase in UV absorbance
- Tm = midpoint b/w transition in absorbance
- stable DNA = higher melting temp
- higher GC (can h-bond with water) = higher melting temp due to base stacking (shifts Tm to right)
Single-stranded RNA
how is stability supported?
(found single stranded most of the time)
- hydrophobic interactions (via base stacking drive structural stability even w/o base pairing)
- driven by VDW and entropic effects
- hairpins can occur from base pairing in same strand
G-U wobble pairing
(general idea)
- normal: GC, AT
- RNA: GU
- U does not have methyl group
angle + distance b/w the ribose C-
1’ bonds do not match A-U or G-C pairs; position of bases are shifted versus the standard base-pairs
base tautomerization
concept & effect? AC vs GT tautomer name?
(different chemicals– not just resonance forms)
- between amino & imino form (by swapping H and DB)
- affects H-bonding and where donor/acceptors are
- AC: amino –> imino (DB out ring)
- GT: lactam (DB out ring) –> lactim
(understand changes in structure + predict bp partner)
imino form of cytosine pairs with adenine
Cytosine deamination
what happens and why is this a problem? how is it resolved?
- (spontaneous loss of exocyclic amino groups)
- cytosine deaminated becomes uracil (C–>U….. NH2 becomes carbonyl)
- imino tautomer attacked by H2O resolving to uracil
- this is why DNA has T instead of U; this rxn occurs often enough to make an issue if U was used in our genetic code
After C>U, DNA rep inserts A across C. Mutation: C>U & G>A (and U>T after more replicatio)
Presence of thymine in DNA instead of uracil
Why?
- bc of spontaneous cytosine deamination (C –> U)
- thymine methyl distinguishes it from C–> U
- uracil in DNA efficiently removed & repaired
Cytosine methylation
purpose? issues when occured with which other process?
- way for cells to mark themselves (cytosine –> 5-methylcytosine)
- no change to H-bonding and genetic info (methyl in same place as thymine methyl in pyrimidine ring)
- problem if deamination (C>U) also occurs (5-Me-C forms T which is normal in DNA and hard to repair)
general transesterification
function? steps of mechanism?
(essentially one chemistry for all backbone changes)
- cutting, extending, & joining nucleic acid sequences
1. nucleophilic attack on a phosphate (OH2)
2. pentavalent phosphate intermediate (phosphate + nucleophile)
3. resolved by diff group leaving besides phosphate (switching of whats bound to make/break bond)
RNA self-cleavage
what happens? why do we have to be more careful with RNA compare to DNA?
what derivative formed?
- 2’-OH on RNA well positioned to attack phosphate and kick off adjacent 5’-O (less stable than DNA)
- spontaneous cleavage is slow, but can be accelerated by heat or basic conditions/cations (2’OH deprotonated to make good nuc)
- self cleavage leads to 2’, 3’ - cyclic monophosphate derivative
Making/breaking backbone bonds
Enzyme functions
nuclease, polymerase, ligase
nuclease (DNA/RNA, ss/ds, nuc seq)
- breaks DNA or RNA backbone (DNase, RNase)
- hydrolysis - transesterification onto water
- exonuclease: cuts ends
- endonuclease: cuts within
polymerase
- joins nucleotides into nucleic acid polymers
- adds to end of existing DNA/RNA
- always adds at 3’ end, on a 3’-OH
- uses nucleotide 5-triphosphates : NTPS of dNTPs
ligase
- joins 3’ end of one polymer to 5’ end of another
(synthesis occurs 5’ > 3’ always)
restriction endo nucleases
cuts only what kind of dna? why?
- cuts middle on dsDNA at specific “recognition sequence” 4-8 bp long
- cuts only unmethylated DNA
- defense mechanism: bacterial genome is methylated; virus (bacteriophage) DNA is not methylated
adding nucleotides to DNA or RNA
Polymerase mechanism
- 3’-OH activated for nuc attack on 1st alpha phosphate of dNTP (or rNTP) with 5’ triphosphate
- inorganic pyrophosphate leaves (PPi)
(alpha phosphate now incorporated into backbone)
(dNTP = DNA molecule with triphosphate group)
(like typical condensation rxn, but PPi leaves instead of H2O)
sanger dideoxy sequencing gel
- ## 3’-H dideoxynucleotide is a chain terminator (no OH to serve as nucleophile)
- four reactions: one per ddNTP; all 4 dNTPs present; trace amounts of ddNTP
- can determine entire sequence by running on separating lanes
gel electrophoresis
- negative nucleic acids migrate towards positive terminal
- gel acts as sieve, smaller molecules moving quicker
- DNA molecules of same size form bands
flourescent ddNTPs
steps?
(modern sanger sequencing)
- different ddNTPs coupled to different flourescent dyes in a single sequencing rxn
1. (like before) ddNTP terminates chain & marks base at end of chain
2. capillary gel electrophoresis used instead of slab gel
3. laser fluorescent detector calls each base as it passes through (shorter pass first)
separated by size, but dye also used
reversible terminator sequencing
- 4 modified dntps used with particular fluorescent label and a 3’ blocking group.
- terminating nucleotide is read,
block is removed, and polymer is extended to read the next nucleotide - disadvantage is that reads are
short, ~150 base pairs
millions to billions of molecules can be sequenced simultaneously.
