Chp 4—Nucleic Acids & Molecular Cloning Flashcards
purines
A
G
pyrimidines
C
T
U
double-ringed bases
purines
how to tell A from G?
A has NH2 next to N furthest from bridge
G has carbonyl next to N furthest from bridge, and NH2 between Ns
how to tell C from T?
C has NH2 directly above R
T has carbonyl directly above R, and methyl on the left side
how to tell T from U?
T has methyl on carbon next to carbonyl
U does not
adenine
guanine
cytosine
thymine
uracil
nucleoside
base + sugar
ribose vs deoxyribose
ribose has 2’ OH
deoxyribose does not
nucleotide
base + sugar + phosphate(s)
phosphate is bonded to…
5’ flag of sugar
names of 3 phosphates on NTP
alpha
beta
gamma
sugar in ATP
ribose
bonds joining nucleotides
phosphodiester bonds
reaction steps for formation of phosphodiester bonds
- free base deprotonates 3’ OH
- (-) oxygen attacks alpha phosphorus on an NTP
- 2 other Ps leave
- strand now has a 5’ end (old) and a 3’ end (new)
2 hydroxyls involved in the phosphodiester bonds
3’ OH
5’ OH
explain phosphodiester bond hydrolysis
in an alkaline environment, free base deprotonates the 2’ OH on ribose
O- attacks nearby phosphorus, breaking the bond with the next nucleotide
H-bonds between A & T
H-bonds between C & G
2
3
chargaff’s rules (4)
- base comp of DNA varies from species to species
- DNA from different tissues have same comp
- base comp does not change with age, nutrition, environment
- A + G = C + T
A + T hydrogen bonds
C + G hydrogen bonds
number of non-canonical base pairings
28
base stacking is via …. interactions
pi-pi
base stacking bond strength
pur/pur»_space; pur/pyr»_space; pyr/pyr
4 contributors to DNA structure
- base pairing; H bonds
- base stacking; pi pi
- syn & anti conformers
- ribose conformers
(anti/syn) conformer is preferred
anti
sugar pucker function
minimizes non-bonded interactions between substituents
common sugar puckers
C2’ - endo
C3’ - endo
Z-DNA conformers
alternating syn and anti conformers along backbone
continuity of DNA replication
semi-discontinuous
helicase
unzips DNA
primase
adds RNA primer
DNA polymerase
binds RNA primer and adds bases
exonuclease
removes RNA primers
ligase
seals replicated DNA together
30-50 divisions
Hayflick limit
3 stages of transcription and translation
- initiation
- elongation
- termination
transcribes DNA to mRNA
RNA polymerase
RNA read…
5’ to 3’
AAs synthesized…
N’ to C’
start codon
AUG
stop codons
UAA
UGA
UAG
translational repression requires…
partial complementarity between miRNA and mRNA
translational degradation requires…
perfect complementarity between miRNA and mRNA
stem and loop shaped RNA
mi RNA
ribosome sites
E P A
5 main base modifications
methylation
deamination
sulfur substitution
base isomerization
double bond saturation
why is there thymine instead of uracil in DNA?
- uracil subject to more modifications
- thymine more expensive to make
- methyl on thymine protects it/makes it more stable
- cytosines deaminate many times a day to give uracil, which could not be corrected if uracil was in DNA already
length of chromosomes
47-247 Mb
avg length of chromosome
4 cm
histone/DNA arrangement models
solenoid
zigzag
protein synthesis steps
- make complementary DNA strand from template
- change Ts to Us to give complementary RNA strand
- flip RNA strand to give a 5-3’ RNA (direction of reading)
- use codon chart to get polypeptide
12 steps of molecular cloning
- amplification of gene via PCR
- digestion of gene with restriction enzymes
- digestion of host vector with restriction enzymes
- isolation & purification of BOTH gene and vector with gel
- sequencing of BOTH with fluorophores
- ligation of gene + vector to give recom.plasmid
- identification of product via gel
- transformation of recom.plasmid into host cells
- selection of host cells carrying plasmid
- isolation & purification of recom.plasmid
- diagnostic digestion & gel
- sequencing of recom.plasmid
steps of PCR
denaturing
annealing
elongation
what not to do do a plasmid with restriction enzymes
cut through the origin or your selection genes
nucleotide + fluorophore
ddNTP
ligation uses…
DNA ligase
PCR uses enzyme…
Taq polymerase
2 methods of transformation
heat shock/chemical
electric shock/electroporation
transduction
virus brings plasmid into cells
transfection
plasmid brought into eukaryotic cells
difficult
2018 Nobel Prize
2020 Nobel Prize
Directed Evolution
CRISPR