Biochemistry Flashcards
(326 cards)
1
Q
how far apart do h bonds hold water molecules apart
A
3A
2
Q
deltaG > 0
A
endergonic reaction, not spontaneous
3
Q
deltaG=0
A
equilibirium
4
Q
deltaG< 0
A
exergonic reaction, spontaneous
5
Q
what happens if you change a protein’s solvent
A
denatures - hydrpphobic effect
6
Q
when can any weak acid or base act as a buffer
A
when ph is almost pka
7
Q
why can amino acids act as buffers at different phs
A
2 different ionisable groups, 2 different pkas
8
Q
zwitterion
A
no overall charge but is ionised
9
Q
ala
A
alanine
10
Q
a
A
alanine
11
Q
arg
A
arginine
12
Q
r
A
arginine
13
Q
asn
A
asparagine
14
Q
n
A
asparagine
15
Q
asp
A
aspartate
16
Q
d
A
aspartate
17
Q
cys
A
cysteine
18
Q
c
A
cysteine
19
Q
gln
A
glutamine
20
Q
q
A
glutamine
21
Q
glu
A
glutamate
22
Q
e
A
glutamate
23
Q
gly
A
glycine
24
Q
g
A
glycine
25
his
histidine
26
h
histidine
27
ile
isoleucine
28
i
isoleucine
29
leu
leucine
30
l
leucine
31
lys
lysine
32
k
lysine
33
met
methionine
34
m
methionine
35
phe
phenylalanine
36
f
phenylalanine
37
pro
proline
38
p
proline
39
ser
serine
40
s
serine
41
thr
threonine
42
t
threonine
43
trp
tryptophan
44
w
tryptophan
45
tyr
tyrosine
46
y
tyrosine
47
val
valine
48
v
valine
49
CORN law
l isomers
COOH, R, H, NH2
50
resonance
partial double bond properties due to sharing electrons between N and O so no rotation around peptide bond
51
what do phi and psi angles describve
shape of proteind
52
ramachandran plot
shows angles around individual alpha carbons
each dot is a pair of phi and psi
shows steric limitations placed on amino acid residues in proteins
53
why is trans conformation preferred
8kj/mol more stable than cis
54
why is trans more stable than cis
steric hindrance
55
why is proline sometimes cis
less steric hindrance
56
what physicochemical interactions determine 3D shape of proteins
salt bridges
h bonds
vdw interactions
hydrophobic interactions
covalent bonds
disulphide bonds
57
charged amino acids
asp
glu
his
lys
arg
58
polar amino acids
asn
ser
the
59
aliphatic amino acids
ala
ile
leu
met
val
60
aromatic amino acids
phe
trp
tyr
61
what can form between oppositely charged side chains
salt bridgeswh
62
which amino acids are ionised at physiological ph
asp and glu
63
what interactions do polar amino acids form
h bonds
64
what interactions do aliphatic a.a form
van der waals
65
when is the attraction of a.a to non-polar atoms maximised
1A apart
66
why are atoms held apart
energetically favourable to be at that distance
67
what do hydrophobic interactions do to bipolar a.a residues
bury non-polar side in core and leave polar outside
68
why do aromatic a.a not appear inside of protein folds
too big
69
why is proline found on extremities of proteins
disrupts backbone H bonding in alpha and beta
70
soluble proteins
in cytosol/ plasma rather than membrane
71
properties of alpha heliux
right handed/ clockwise
no free space inside
side chains point outwards
72
alpha helix favoured residues
met, ala. glu, lys
73
alpha helix unfavored residues
pro, gly, asp
74
h bonding in alpha heliux
every carbonyl o forms h bond with amide h 4 residues along
75
pitch
how far it goes up every time it goes round
76
coiled coils
stripes of amino acids wrap around each other on different helices
77
3 10 helix
every 3 residues
78
pi helix
every 5 residues
79
beta sheets properties
h bonds between adjacent backbones
antiparallel favoured as h bonds perfectly perpendicular
80
beta barrel
entirely made of beta strands joined by h bonds
81
example of beta barrels
retinol binding protein
green fluorescent protein
82
loops and turns
most variable and biologically active parts of proteins
83
reverse turn beta hairpin
links beta strands
84
greek key motif
fold beta hairpin over 2 beta strands
85
effect of proline on secondary structures
forms kinks in alpha helices
do not form beta sheets
86
non-allosteric interaction
protein binds one ligand
P+L<>PL
87
allosteric interaction
protein binds multiple ligands
changes affinity
P+LA+LB<>PLA+LB<>PLALB
88
Hb structure
2 alpha chains, 2 beta chains
89
HbF structure
2 alpha chains, 2 gamma chains
90
91
alpha chain
141 a.a
92
beta chain
146 a.a
93
prosthetic group
non a.a group
94
Mb structure
one chain - 154 a.a
one haeme group
similar 2 and 3 structure to hb
95
haeme group
porphorin ring sitting in hydrophobic cavity
distal and proximal his
96
what stabilises the distal his in haeme
o2 forms h bonds with it
97
P+L<>PL
what do the forward and reverse reactions represent
ka
kd
98
P+L<>PL
what is the velocity of the forwards reaction (vf) dependent on
ka
[P] and [L]
99
P+L<>PL
what is the velocity of the reverse reaction (vr) dependent on
kd
[PL]
100
mass action at equilibrium
[P][L]ka = [PL]kd
101
caclulaction for
dissociation constant
kd/ka = Kd = [P][L]/[PL]
102
fractional occupation equation
Y = [L]/Kd+[L]
103
low [L]
low binding site occupation
104
[L] = Kd
half of binding sites are occupied
105
[L] much greater than Kd
most binding sites are occupied
106
when does Kd differ
if protein has multiple possible ligands with different affinities
107
Kd equation for myoglobin
[deoxyMb]*[O2]/[oxyMb]
108
what is Kd substituted for with gases
p50
109
p50 equation for myoglobine
[deoxyMb]*p50/[oxyMb]
110
p50
partial pressure to fill 50% of binding sites
111
fractional occupation equation in terms of o2
Y=pO2/P50+pO2
112
113
rank body systems in descending order in terms of po2
lungs
resting tissues
exercising tissues
114
Tense form
deoxyhaemoglobin
115
what affinity for o2 does the tense for have
low
116
relaxed form
oxyhaemoglobin
117
what affinity does the relaxed form have
high
118
what does the sigmoidal curve for haemoglobin show
positive binding cooperativity
binding of one o2 increases affinity for other sites
119
hill coefficient = 1
no cooperative binding
120
hill coefficient >1
positive cooperativity
121
does the t or r state have a higher p50
t state
122
how does equilibrium shift as more o2 bind
favours the r state
123
2,3-bpg
allosteric effector
binds to T state more tightly than R state
increases p50
124
the Bohr effect - H+
H+ as allosteric inhibitor
binds preferably to T state
125
the Bohr effect - co2
co2 binds directly to N terminal groups of Hb forming carbamate
reduces affinity for O2
126
importance of purifying proteins
prevent interference with experiments
remove proteins with related activity
impure proteins resistant to forming crystals in x-ray crystallography
127
advantages of using prokaryotes to produce proteins
very easy to manipulate genome
easy to grow in large cultures
high yield
128
disadvantages of using prokaryotes to produce proteins
different post translational modifications to mammals
poor folding of complex proteins
129
advantages of using unicellular eukaryotes to produce proteins
easy to manipulate genome
easy to grow in large quantities
high yield
mammalian like post translational modifications possible
130
disadvantages of using unicellular eukaryotes to produce proteins
moderate ability to produce more complex proteins
131
advantages of using cultured mammalian cells to produce proteins
full range of post translational modifications
can fold complex proteins
132
disadvantages of using cultured mammalian cells to produce proteins
difficult to genetically manipulate
hard to grow in large quantities
poor yield
very expensive growth medium
133
assay method
add sample of enzyme to substrate
mix and follow absorbance in spectrophotometer
134
liquid phase of column chromatography
solution containing protein mixture
135
stationary phase of column chromatography
porous solid matric
136
column chromatography: gel filtration
separates based on size of protein
137
column chromatography: ion exchange
charge of protein
138
column chromatography: hydrophobic interaction
separates based on hydrophobicity
139
column chromatography: affinity chromatography
separates based on protein interactions
enzyme-substrate
antibody-antigen
140
how does gel filtration chromatography work
carb polymer beads
small molecules enter aqueous spaces within beads
large molecules cannot enter beads
141
how does ion exchange chromatography work
medium has a permanent charge
protein has many charged amino acids
142
isoelectric point
pH at which protein has no overall charge but is ionised
143
how does charge change as pH becomes more basic
overall charge decreases depending on numbermof ionisable side chains
144
how do you alter what proteins bind to the column
change pH
145
how to eliminate proteins from ion exchange column
change pH
increase buffer's salt concentration
stepwise or gadient
146
does stepwise or gradient have a higher resolution
gradient
147
SDS-PAGE protein purification
based on size
gel gives proteins negative charge
voltage passes through gel - makes positive
heavier proteins don't move as dark as as lighter proteins
148
examples of co-enzymes/cofactors
metal ions, NADH, haem
149
where are hydrophobic amino acids found in enzymes
inside
150
similarities of enzymes to chemical catalysts
catalyse reaction without changing
reaches equilibrium faster but does not shift equilibrium
151
differences of enzymes to chemical catalysts
high substrate specificity
high catalytic power
require less intense conditions
152
Kcat
number of molecules a single enzyme can bind and convert to product every second when substrate is not a limiting factor
153
how is enzyme activity determined
assay measuring
rate of product formation
rate of loss of substrate
rate of production of cofactor
154
equation to calculate enzyme activity
d[P]/dt = -d[S]/dt
155
why must initial rate be calculated
substrate used up in reaction reduces rate
156
enzyme activity unit
amount of enzyme which transforms 1 micromole of substrate per minute at 25 degrees
157
specific activity
number of enzyme units per mg of protein - purity
158
Vmax
maximum rate
159
Km
Michaelis constant
measure of affinity for a substrate
160
line weaver-burk plot
reciprocal of m-m plot
161
y=mx+c of line weaver burk
1/V = Km/Vmax * 1/[S] + 1/Vmax
162
irreversible enzyme inhibit
covalent bonding to active site
163
competitive reversible enzyme inhibitors
bind to active site
overcome at high [S]
Vmax same but Km increases
164
non-competitive enzyme inhibitors
bind to allosteric site
Vmax reduces, Km same
165
how does lineweaver burk plot change with inhibitors
gradient becomes steeper
166
why can't we perform acid-base catalysis experimentally
not possible to have 2 pHs at once
167
how does RNase perform hydrolysis
His 12 acts as base
His 119 acts as acid in first step
reverse in second step
substrate steered into active site by oppositely charged residues
168
what contributes to the catalytic activity of RNase
enhanced reactivity of side chains
orienting of substrate wrt catalytic groups of enzyme
169
why is L-lactate dehydrogenase stereospecific
Arg 109 angles carbon down to ensure L-isomer produced
170
chymotrypsin
c-terminal side of bulky hydrophobic and aromatic amino acids
171
trypsin
c-terminal of K or R
172
elastase
c-terminal side of small amino acid
173
mechanism of identifying enzyme's catalytic triad
nucleophilic attack - his acts as base
acyl-enzyme intermediate - His acts as acid
substitution of tetrahedral intermediate
general base catalysis
174
specificity pocket
orients peptide bond for cleavage by catalytic triad
175
zymogen
inactive protease which must be cleaved to become active
176
how are serine proteases regulated biologicallty
zymogens secreted from pancreas to duodenum
trypsin activated by enteropeptide in duodenum
trypsin inactivated by pancreatic trypsin inhibitor
177
assumptions of the fluid mosaic model
proteins at low concentrations
constant thickness
lipids are all the same
178
truth of fluid mosaic model
many protein complexes
many different types of lipids
bilayer constantly changes shape to match protein
all membranes are different
179
amphipathic
hydrophobic and hydrophilic regions on the same molecule
180
properties of cholesterol
polar OH group so slightly amphipathic
controls membrane fluidity and packing
181
why is the membrane asymmetric
each monolayer has a different lipid composition
many cytosolic proteins bind to specific lipid head groups
182
lipid movement
lateral diffusion
hydrocarbon chains are flexible and dynamic
lipids rotate freely around their vertical axis
183
properties of rigid gel phase
occurs at low temperatures
restricts lateral diffusion
transition temperature is lower with shorter chain hydrocarbons
184
evidence for lateral diffusion
different lipids labelled with fluorescent markers
after 40 minutes they had integrated
conclusion: free diffusion of cell surface proteins with hybrid membrane
185
properties of integral membrane proteins
embedded in bilayer
bound to membrane by hydrophobic forces
can only b e separated from membrane using disrupting agents
single or multipass
insoluble in aqueous buffers
186
properties of peripheral/ extrinsic proteins
bound to surface by h bonds or salt bridges
easily dissociate from membrane under mild conditions
can be anchored to lipids
187
what is unique about membrane proteind
either wholly helix or wholly sheet
188
physical chemistry of membrane proteind
many hydrophobic amino acid
present hydrophobic surface to acyl chains
difficult to study outside of membrane environment
189
properties of alpha helices in the membrane
thermodynamically stable
all h bonds are satisfied
hydrophobic residues face the acyl chains
190
properties of beta sheets in the membrane
alternate polar and hydrophobic aa
hydrophobic residues face bilayer core, polar residues face interior
intra-chain h bonds between strands
191
transporter
down or against conc gradient
192
channel
only allow diffusion down conc gradient
193
na+/k+ ATPase
actively exports 3Na+ and imports 2K+ using 1 ATP
inward sodium gradient
negative delta G
free energy used to drive transporters
194
prokaryotic membranes
electron transport chain generates proton gradient
drives ATP synthase
195
genome
all genetic information of an organism
196
gene
basic unit of inheritance
197
difference between ribose and deoxyribose
ribose has OH on the 2' carbon
198
purines
double ring structure
A&G
199
pyrimidines
single ring structutre
C, U & T
200
why is RNA unstable
2' OH acts as nucleophile to break phsophodiester link
201
char gaff's rule
pyrimidine:purine = 1:1
%C=%G, %A=%T
202
why do bases stack
hydrophobic interaction
203
denaturation
double strand to single strand
204
annealing
single strands to double strands
205
minor groove properties
1.2nm
narrow and deep
206
major groove properties
2.2nm
wide and shallow
207
persistence length
length of DNA along which a thermally excited bend of 1 radian occurs
208
why is short DNA stiff
electrostatic repulsion of phosphates pushes against bending
energetically favourable that bases are stacked nicely
209
protein-DNA interactions
proteins bind and recognise specific DNA sequences
recognise dna damage
bind DNA non-spefifically]
210
dNTP
deoxyribo nucleotide triphosophate
211
DNA polymerase 3
main replicating enzyme
9 subunits
250-1000 nucleotides per second
3'-5' exonuclease activity
proofreading
212
DNA polymerase requirements
dNTPs as precursors
can only add dNTP to 3' end of nucleic acid
3' primer
magnesium ion
213
initiating DNA replication
initiator binds to origin
easily melted A&T nucleotides separated
DNA helices binds to break more H bonds
214
ssDNA binding proteins
prevents reannealing of sDNA during replication
DNA polymerase removes them as it goes along
215
properties of primase
synthesises RNA primers
binds to 3' hydroxyl
no proofreading
no specific initiation sequence
frequency of priming is different in leading and lagging strands
216
how often does replication restart in the lagging strand
~1000 bases
217
direction of DNA replication
5' to 3'
218
purpose of mg ion in polymerase catalytic site
stabilise the phosphate
activate OH to make it a better nucleophile
219
requirements for polymerase catalytic site
asp residues
mg ion
triphosphate
220
nuclease
cleaves phosphodiester bonds
221
exonuclease
ends of nucleic acid
222
endonuclease
within nucleic acid
223
prokaryotic connecting Okazaki fragments
polymerase 1 removes primers and replaces with DNA
3'-5' exonuclease rpoofreading
DNA ligase catalyses phosphate linkage
224
terminating replication
tus binds to terminating sequence
physical block to replication fork
225
direct repair
specific base damage
removed directly by enzyme
226
mismatch repair
incorrect bases
227
base excision repair
range of damaged bases
228
nucleotide excision repair
wide range of bulky DNA damage
229
RNA secondary structures
helices
hairpins
bulges
230
RNA base interactions
AU, GU, AUA triple
231
prokaryotic RNA polymerase requirements
all dNTPs
3' hydroxyl to attach dNTPs
starts at A or G
promoter DNA and sigma unit to initiate
232
bacterial promoters properties
where RNA polymerase starts transcription
define which strand is copied
requires sigma factors
different strengths
233
promoter strength
stronger promoters produce more proteins
234
promoter unwinding
positions new nucleotide into the active site of RNA polymerase
defines where transcription starts
235
rho-dependent transcription termination
occurs at specific sequences
binds to rut sites in transcription
pulls RNA out of RNA polymerase so RNAP falls off
236
rho-independent trasnscription termination
occurs at specific sequences
237
transcription inhibitors
rifampicin
binds to beta subunit of bacterial RNAP
prevents initiation but not elongation
blocks path of elongation at 2-3nt length
238
transcription inhibitors
actinomycin D
intercalates into DNA
prevents initiation and elongation
can also interfere with replication
239
which amino acids to pyrimidines generally code for
hydrophobic
240
which amino acids to purines generally code for
hydrophillic
241
silent or synonymous mutations
same Amino acids
242
missense or non synonymous
changes 1 amino acid
243
nonsense or stop
truncated protein
244
frameshift
scrambled protein structure
245
properties of tRNA
small, 74-93 nucleotides long
folds from clover to L shape
246
wobble position
1st and 2nd codons bind to tRNA normally but 3rd is less constrained
247
activation of amino acids
catalysed by amino acyl tRNA transferases
aa added to ATP releasing 2Pi
aa added to tRNA forming amino acyl tRNA
releases AMP
248
why must amino acids be activated
peptide bond formation between free amino acids is unfavourable
249
can ribosomes check aa
no
250
amino acid recognition
synthetases are highly specific
correct aa has highest affinity for active site of synthetase
251
tRNA recognition
synthetases must recognise correct tRNA
structurally and chemically complimentary
252
aa proofreading
after initial attachment, aa forced into editorial site
only incorrect aa fit
once in, hydrolysed from tRNA
253
peptide tRNA
bound to mRNA and had polypeptide chain attached
254
amino acyl tRNA
free with next amino acid in chain attached
255
prokaryotic 50S component
Mr 1.6 million
rRNA and proteins
256
prokaryotic 30S component
Mr 900k
rRNA and proteins
257
initiation of translation in prokaryotes
5'-3' at AUG or GUG codon
30S component binds to ribosome binding site and places AUG in active site
IF 1,2 and 3 + GTP allow 50S component to bind
forms 70S initiation complex
258
elongation of translation in prokaryotes
amino acyl tRNA binds to empty A site
proofread and EF-Tu dissociates
peptide transferase reaction
large subunit translocation
small subunit translocatiom
259
explain proofreading during elongation of prokaryotic translation
GTP hydrolysed
incorrect tRNA dissociates
260
what escorts tRNA to the A site
EF-Tu
261
describe the peptide transferase reaction
bond to peptidyl tRNA broken
chain transferred to amino acyl tRNA
synthesis starts at N terminus and new amino acid is added to C terminus
catalysed by ribozyme
262
ribozyme
23S rRNA
263
explain large subunit translocation
large subunit moves forward
stabilised by EF-G
GTP binds to A site
energetically unfavourabke
264
explain small subunit translocation
GDP released
small subunit moves to next codon
energetically favourable
265
termination in prokaryotic translation
release factor binds to stop codon in A site
C terminus hydrolysed and protein released
266
why does the release factor cause ribosome to detach
ribosome changes conformation so subunits detach and mRNA is released
267
why are prokaryotic transcription and translation coupled
dna and ran are both in the cytosol
268
antibiotics properties
stall initiation
prevent elongation
induce miscoding
269
basal transcriptional activity
genes are always on unless controlled
strength of promoter dictates gene expression
270
genetic switches
repressors or activators
proteins that bind to specific dna sequence controlled by binding of a ligand
271
repressors
bind to operators
cause RNA to detach
272
operator
overlaps with RNAP binding site
273
activators
bind to sites which do not overlap with RNAP binding site
helps RNAP bind to promoter
274
purpose of the lac operon
low glucose but still need to synthesise ATP so uses lactose
275
the lac repressor
RNAP binds in the presence of lactose
releases lac repressor from operator
276
the CAP activator
CAP detects cAMP
CAP binds to operon, cAMP binds to CAP
RNAP binds
277
when are high levels of cAMP present
low levels of glucose
278
lac operon when glucose is present and lactose is not
expression of lac operon repressed
279
lac operon when glucose is not present but lactose is
lac operon expressed
280
no glucose or lactose in lac operon
expression repressed
281
glucose and lactose present in lac operon
expressed
282
lamda repressor process
lamda repressor binds to OR1
at high concentrations more lambda repressor binds to OR2
at higher concentrations binds to OR3 SO RNAP cannot bind
cro repressor blocked
283
why does lama repression fail
DNA damage so proteolysis of lambda repressor
cro repressor promoter no longer blocked
284
cro repressor
RNAP binds to cro promoter producing cro repressor
lamda bacteriophage genes expressed
lytic state reachedq
285
lamda repressor high, cro low
lysogenic state
286
lamda repressor low, cro high
lytic state
287
size of DNA double helix
2nm
288
size of beads on a string form
11nm
289
size of chromatin fibre of nucleosomes
30nm
290
why can we not be sure of 30nm fibre
never actually been observed in the cell
may only exist due to extracellular environment
291
size of chromatin fibre folded into loops
700nm
292
size of mitotic chromosome
1400nm
293
structure of histone
2 copies of 8 proteins
294
chromatin remodelling enzymes
use energy from hydrolysis of ATP
295
ISW1 enzyme
slides nucleosome along DNA
296
SW1/ SNF enzyme
removes nucleosomes from DNA
297
histone acetyltransferase function
relaxes chromatin via acettylation
298
histone deacetylase function
condenses chromatin
299
differences of eukaryotic DNA replication to prokaryotic genes
during S phase
slower DNAP
multiuple origins of replication as multiple chromosomes
end replication problem solved by telomerase
300
differences of eukaryotic transcription to prokaryotic transcriptiuon
RNAPs require accesory factors for each stage in the cycle
promoters are more complex
RNAPs transcribe through chromatin
301
why does pre-mRNA require processing
unstable
cannot leave nucleus
cannot bind to ribosome
302
why can only RNAP2 transcripts be processed
1&3 do not produce translatable mRNA
303
5' capping process
M7G linked through onverted 5'-5' triphosphate bridge to initiating nucleotide of a nascent script
304
why is 5' capping important
prevent RNA degradation by exonucleases
allows transport from nucleus to cytoplasm
initiates translation
recruits splicing factors
305
mRNA splicing
introns removed by spliceosome complex
306
why is splicing useful
different protein combinations can be produced by combining different exon sequences
307
polyadenylation process
ployA tails added to mRNA at the end of transcription by poly-A polymerasew
308
why is polyadenylation useful
protects RNA from degradation
transport of RNA from nucleus to cytoplasm
assists action of ribosome
termination
309
preparing DNA for recombination
add phenol and centrifuge
aqueous layer is DNA and RNA, phenol layer is protein
add ethanol
DNA precipitate left
310
source of restriction endonucleases
bacteria
311
restriction endonuclease recognition sites
specific 4-8bp palindromic sequences
312
activity of restriction endonucleases
cut dsDNA
leaves 5' sticky ends or blunt ends
313
digestion frequency
how often RE cuts
4^n
4 = number of bases
n = length of recognition sequences
314
DNA ligation
covalent bonding of fragments to each other
complementary base pairing forms H bonds
315
process of DNA ligation
T4 DNA ligase joins nucleotides
uses 2ATP
produces 2 AMP and 2 Pi
both gaps closed
316
how to produce a viral vector
replace lamda DNA with foreign DNA
produce viral assay
317
how to produce bacterial plasmid vector
many unique RE sites to insert foreign DNA
318
cloning eukaryotic genes using reverse transcriptase
synthesises cDNA using oligo(dT) primer which binds to poly-A
RNA/DNA hybrid forms
RNAse H digests RNA
ssDNA forms hairpin which primes cDNA
DNAP added to form DNA
S1 nuclease opens hairpin
319
in-vitro DNA synthesis in a tube
ssDNA primer + 4 dNTPs + ssDNA template + Taq DNA polymerase
heat to 95 degrees to break H bonds
320
Taq polymerase properties
heat resistant but prone to error
no proofreading
321
PCR for gene cloning
not dependent on RE sites
greater specificity
322
PCR for viral screening
highly sensitive
shows virus before symptoms appear
323
PCR for forensics
DNA fingerprinting
familial linkage
324
sanger DNA sequencing
dideoxyribonucleotides
determine order of bases
screen mutations of variants
validates PCR
325
Sanger's method
template + primer + dNTPs + 35-S-dCTP + Taq DNAP
tubes have different dNTPs
each reaction terminates at a different base
add products to respective lane of gel
electrical field
read manually 5'-3'
326
improvements to sanger's method
flourescent dNTPs over radioactive
performed in a single tube
