bacterial genetics Flashcards
(300 cards)
1
Q
polymorphism
A
cite in a gene sequence displaying variation in a population
2
Q
hox genes
A
group of related genes controlling body plan of an embryo along a head-tail axis
3
Q
scabrous
A
ethanol reward as memory aid insignalling pathway of long-term memory
4
Q
hangover
A
ethanol tolerance aid as well as response to heat and oxidative stress
5
Q
syntaxin
A
mutations affect ethanol tolerance to sedation, encoding synaptic vesicle release protein
6
Q
simple/ single gene defect disease
A
Duchenne MD
huntington’s
7
Q
complex/multi gene defect disease
A
Cancer
diabetes/obesity
8
Q
wild type
A
unmodified isolate of a species, well-characterized in the lab
9
Q
mutant
A
differs to wild-type in that changes to DNA sequence have occurred
10
Q
mutation
A
specific change/changes to a DNA sequence different to the wild-type
11
Q
phenotype
A
observable trait
12
Q
genotype
A
nucleotide sequence of a region of DNA
13
Q
why are bacteria used as genetic models?
A
much is known about them
easy to manipulate
short generation time
14
Q
vertical gene transfer
A
changes in DNA passed on to progeny
15
Q
horizontal gene transfer
A
changes to DNA passed on from other bacteria/viruses
16
Q
genetic transformation scientists
A
Fred Griffith
- conversion of non-pathogenic strept. pneum into a pathogenic
17
Q
conjugation scientists
A
Lederburg and Tatum
- 2 e coli strains mixed and progeny isolated with both characteristics
18
Q
transduction scientists
A
Zinder and Lederburg
- displayed bacterial viruses carrying DNA from one bacterium to another
19
Q
Bacterial transformation
A
ability of bacterial cell to uptake DNA from other cells in the same environment
20
Q
Competence
A
correct physiological state for recipient cell in order to uptake DNA
naturally/ artificially induced
21
Q
purpose of transformation
A
increase functional/ metabolic capabilities to compete in environment
22
Q
bacterial conjugation
A
gene transfer via cell-to-cell contact mating
23
Q
conjugation mediation
A
conjugative plasmid can induce pilus/ transfer DNA
24
Q
high frequency recombination strains
A
can transfer part of host chromosome to recipient
25
mob genes
allow for genetic transformation of a non-conjugative bacterial cell
26
stages of bacterial conjugation
donor cell attaches to recipient cell via pilus
pilus contracts
1 plasmid DNA strand attaches
recip and donor cell synthesize complementary strands to form an F+ cell
27
bacterial transduction
gene transfer via bacterial virus mediation
28
transducing particle
phage filled wiyh host and chromosomal DNA
29
stages of transduction
1. bacteriophage injects DNA
2. phage enzymes degrade host DNA
3. cell produces more phages incorporating host/ phage DNA
4. transducing particles transform other cells as donor DNA incorporated into recipient chromosome via recombination
30
transcription
decoding genetic info enmcoded by DNA into RNA
1 step process
31
decoding genetic info to proteins
2 step process (transcription/translation
32
bacterial genetic decoding
transcription and translation coupled
genes of related function often clustered on chromosome
33
transcription mediator
RNA polymerase
34
transcription 3 stages
initiation, elongation, termination
35
types of rNTPs
rATP, rUTP, rCTP, rGTP
36
Inititiation
RNA polymerase attaches to promoter sequence in DNA
37
elongation
RNA polymerase moves along template strand, decoding to RNA
38
termination
RNA polymerase recognizes terminating sequence and finishes transcription prior to strand separation
39
1 residue
1 amino acid= 3 ribonucleotides= 1 codon
40
number of possible codons per amino acid
64
41
number of proteins potential depending on reading frame
3
42
number of reading frames
2 strands with 3 proteins each
43
how is choice of reading frame determined
an ATG/AUG start codon
- ribosome binding site in front is complementary to 3' end of 16S rRNA
44
2 stages of bacterial DNA replication
binary fission
DNA synthesis
45
DNA polymerase action in bacterial DNA replication
creates phosphodiester linkage between 5'OH end of DNA and 3'PO4 end on dNTP
46
DNA synthesis precursors
deoxynucleoside triphospates
dATP dGTP dCTP dTTP
47
bacterial replication
begins at oriC before proceeding bidirectionally (creating 2 replication forks) and finishing at terminus C (terC)
48
6 types of mutation
base-pair changes
deletions
inversions
insertions
frame-shifts
duplications
49
2 types of base-pair change mutations
transition/ transversion
50
base pair transition
pyrimidine> pyrimidine/ purine> purine
51
base pair transversion
pyrimidine<>purine
52
3 potential consequences of base-pair mutations
silent mutation (same sequence)
missense mutation (different sequence)
nonsense mutation (stop codon)
53
mutation frequency formula
m/N
no. mutants/ no. bacteria
54
mutant selection
general selection
1. isolate randomly distributed mutants
2. screen
55
specific selection
every isolated mutant of interest screened
56
negative selection
selects against muatnt growth
57
enrichment
negative selection use to promote growth of mutants and inhibiting wild-type/ competition growth w antibiotic
58
positive selection
selective conditions to promote mutant growth
(usually in resistance to a phage)
59
genomics
acquisition, storage, retrieval and analysis of DNA sequence
60
genome size range
(most common?)
0.13-14MBp
most common =3 MBp
61
E.Coli model strain
characteristics?
K12 strain
rapid growth, simple nutritional requirements
62
K12 relatives
meningitis
UTI
gastroenteritis
typhoid
plague
63
E.Coli chromosome size
4.6MBp
64
streptomyces coelicolor chromosome
linear chr
65
parts of streptomyces coelicolor chromosome
core (maintenance genes)
arms (variable extras)
66
ARTEMIS
visualisation tool displaying annotation files graphically
67
metagenomics
study of genetic material recovered directly from environmental samples
68
recombinant DNA industries
food, pharma, agriculture, medical research
69
food recombinant DNA examples
chymosin (cheese manufacture)
golden rice (enzymes promoting B carotene synthesis)
70
pharma recombinant DNA examples
human insulin (pig replacement)
HGH (cadaver replacement)
blood clotting factor VIII (haemophilia)
Hep B vaccine (yeast cell production)
71
medical research recombinant DNA examples
HIV antibody test
72
agriculture recombinant DNA examples
proteins (herbicide resistance)
glyphosphate (herbicide round-up)
73
resources for recombinant DNA tech
enzymes, DNA/ RNA, vectors, cells
74
types of enzyme used in recombinant DNA tech
restriction
taq polymerase
ligase
reverse transcriptase
75
restriction enzymes
cleave DNA at specific sequences
often recognise palindromic 4-8 Bp
76
DNA ligase
anneals
77
taq polymerase
amplification of DNA fragments
78
reverse transcriptase
converts RNA back to DNA
79
2 types of cleavage pattern
symmetrical (blunt end production)
asymmetrical (sticky end production)
80
2 types of DNA in recombinant DNA tech
insert/ vector
81
vector DNA
unique restriction sites
efficient oriC
gene expression regulatory sequences
82
plasmid size
2-200 kbp
83
vectors for larger DNA fragments
bacteriophages
cosmids/phagemids
84
cosmids/ phagemids
genetically engineered hybrids replicating as plasmid and packaged as bacteriophage
85
clone production steps
1. prepare insert/vector
2. ligate both
3. transform recomb DNA into host
4. select hosts containing DNA
86
isolation of specific inserts
cleaved via restriction nuclease
87
amplification of non-specific insert
cDNA copies total mRNA and reverse transcriptase used
88
amplification of specific DNA
PCR
89
Steps in DNA insertion
1. isolate insert from RNA/DNA
2. ligate insert into vector
3. transform into host cell
4. select recomb DNA hosts
90
ligation of insert into vector
cleaving of plasmid and ligase annealing of insert
91
transformation into host cell
1. plasmid mixed in
2. heat shock and CaCl induces competence of cells
3. taken up by plasmid and selectively cultured
4. recomb purified and expressed
5. expression in host
92
selection of recomb DNA hosts
selective medium growth
93
insertional inactivation
DNA fragment in polylinker disrupts lacZ gene > inactive b galactosidase > no blue pigment detected by X-gal
94
how to check for recombinant DNA
Hybridization of ssDNA to probe
screening for protein expression
PCR
95
E.coli requirement for expression in host
expression vector
96
expression vector mechanism
contains promoter sequence recognised by host RNA polymerase and therefore expressed gene ligated at 3' end
97
direct expression of recombinant protein
purification
investigation of protein function
98
expression of modified version of protein
cloned gene engineered to : change protein properties/ investigate fine details of a protein
99
non-coding DNA
introns
exons
100
intron
nucleotide sequence within gene
removed via splicing during RNA maturation
101
exon
gene region produced after intron removal
102
when would inactive form be expressed
if genomic DNA ligated into expression vector
103
bacterial expression systems advantages
simple
cheap
short generation time
large yield
104
bacterial expression systems disadvantages
can fail to fold crrectly, losing bio activity
toxic proteins to bacterial cell
no post-trans modifications
105
yeast expression systems advantages
simple
cheap
resembles mammalian cells
quick
pos trans modifications
106
yeast expression systems disadvantages
protease containing (degrades recomb proteins)
differing post-trans modifications
107
insect cell expression systems advantages
cheaper than mammalian
high-level expression
correct folding
post-trans modifications
108
insect cell expression systems disadvantages
post-trans modifications differ from mammalian cells
109
mammalian expression systems advantages
best for mammalian
correct folding
post trans modifications
110
mammalian expression systems disadvantages
expensive
complex cells
grow to lesser densities
111
example of expression for commercial use
diabetes
112
example of protein expression for further research
HIV
113
where's insulin produced
beta cells of islets of langherhan in pancreas
114
bovine/porcine insulin considerations
side-effects
ethics
purification issues
contamination issues
115
insulin structure
2 polypeptide chains linked by disulfide bonds
116
synthesis of insulin procedure
folding stabilized by disulfide bonds, connecting peptide removed
117
recombinant insulin production
proinsulin gene (2introns) coding for mRNA which is reverse transcribed to cDNA, inserted into a recombinant plasmid and transforms bacterium
118
genetic model organisms wanted characteristics
short lifespan
readily available
amenable to genetic transformation
large offspring yield
small;
rapid development rate
119
e.coli cell
gram-negative, rod-shaped bacterium
120
generation time of E.coli
20-30 mins
121
e coli advantages
simple
safe
short generation times
122
e coli disadvantage as model organism
prokaryptic so differ to eukaryotic
123
homologue
gene related to another gene via descent from a common ancestoral DNA sequence
124
orthologue
genes from different species evolved from a common ancestral gene
125
paralogue
genes generated by a duplication event
126
gene knockout
gene sequences completely/ partially removed, inhibiting gene expression
127
gene knockdown
techniques interfering/ reducing gene expression
128
yeast budding/fission advantages
share characteristics of cell div cycle, gene expression, signalling pathways w humans
short life cycle
small
simple growth/ storage
easily transformable w plasmids
trivial construction of gene knockouts/ knockdowns
129
drosophilia melanogaster advantages
small
easy to collect
2 week life cycle
14000 gene encoding
easy to cross
phenotypic markers
transformable
research useful
130
p transposon
jumping gene used to transform drosophilia
131
drosophilia research areas
cell signalling
development
neurological disease
132
disease model
mutant model organism mimicking human phenotypes of disease
133
spinal muscular atrophy
neurodenegerative disease decreasing motor function
caused by mutation in smn gene
flys hold orthologous smn gene
134
alzheimers
accumulation of amaloid beta peptide
progressive neuron loss
135
C. elegans
small, non-parasitic nematode
transparent
easy to manipulate
both sexes
300 offspring pp
2-3 week lifespan
136
c. elegans dsRNA interference procedure
1. dsRNA complementary to region of interest introduced
2. process to short interfering siRNA's
3. prevtnion of expression of region of interest
137
zebra fish
transparent embryos
200 eggs weekly
difficult to sequence etc
knockdown possible w morphilinos
micro-injectible
138
zebra fish research areas
vertebrate development
neurobiology
toxicology
disease/ drug discovery
139
mus musculus
closely related to humans
identical gene composition offspring (lab inbreeding)
embryonic manipulation
knockouts available
140
mouse knockout process
1. embryonic stem cells w one copy of target cell deleted
2. injected into early embryo
3. hybrid embryo introduced to mouse
4. hybrid offspring
5. further cycles > homozygous KO
141
C value paradox
gene number doesn't increase linearly with genome size
142
percentage of human genome-wide repeats
44%
143
G-banding
mild proteolyis then GIEMSA
AT rich
144
R banding
heat denaturation then GIEMSA
GC rich
145
Q banding
quinacrine stain
AT rich
146
C-banding
BaOH then GIEMSA
Constitutive heterochromatin
147
alpha satellite
large arrays of repeated sequences
148
assembly site for kinetochore
centromere
149
positioning of centromere
metacentric
submetacentric
arcocentric
telocentric
150
short chr arms
p
151
long chr arms
q
152
telomere
specialised region at chr ends
153
function of telomere
allows distinguishment between real chromosome end/ unnatural so that unnatural is destroyed
end replication problem
154
end replication problem
3' terminal isn't copied > ssDNA overhang degraded
telomeres therefore prolong replicative senescence
155
telomerase
recognizes uncopied region
adds multiple copies of 'TTAGGG' repeat
replication machinery synthesizes other strand
156
histones
form octamer which DNA wraps to form nucleosome
157
euchromatin
relatively uncondensed
associated w active genes
158
heterochromatin
condensed
associated w silenced gene-poor regions
159
B-chromosomes
additional chromosomes possessed by some in population
160
holocentric
no singular cetromere, multiple kinetochores throughout
161
kinetochore
protein structure located at centromere, serving as attachment point for mitotic spindles
162
centriole
9 microtubule groups, generating mitotic spindle fibres
163
G2 of interphase
chromosomes extended
chromatin duplicated
164
chiasma
physical connection between 2 non-sister chromatids
leads to crossing over between pairs
165
meiosis prophase 1
leptopene
zygotene
pachytene
diplotene
diakenesis
166
synaptonemal complex
nucleoprotein zipper between paired homologous chromosomes
role in crossing over/ chiasma formation
167
disjunction
1/2 of tetrad migrates to each pole
168
oocytosis
begins in embryonic ovary and arrests in prophase 1
169
nondisjunction
aneuploid gametes
170
trisomy
3 copies of specific chromosome
171
mitosis vs meiosis
meiosis is reductional division
cant occur in haploids
2 successive divisions
mitosis 1 division
occurs in haploids/ diploids
172
sexual reproduction disadvantages
advantages
time and energy to find mate
potential breaking apart of favourable gene
better equipped for environmental changes
173
2 hypotheses of sex determination
protenos mode
lygaeus mode
174
heterogametic
producing unlikely gametes
175
homogametic
producing uniform gametes
176
klinefelter syndrome
tall with feminised physique
poor beard growth
low IQ
breast development
female pubic hair
testicular atrophy
osteoperosis
44 autosomes + XXY
177
Turner syndrome
short
constricted aorta
elbow deformity
no menstruation
brown spots
44 autosomes + 1X
178
human Y chromosome regions
PAR
SRY
MSY
PAR
179
MSY regions
euchromatin
centromere
euchromatin
heterochromatin
180
PAR
pseudoautosomal regions
sharing homology with X chromosome
synapse and recombine with x chromosome in meiosis
181
MSY
Male Specific Region of Y
doesnt synapse w x chromosome
182
SRY
sex determining region of Y
produces testes determining factor,triggers undifferentiated gonadal tissue of embryo to form testes
183
dosage compensation
females likely produce twice as much gene product for all X-linked genes
> mechanism required to equate X-linked gene product doses
184
Barr body
condensed X-chromatin
inactivated X chromosome
185
barr body formation
dosage concentration supported by X chromosome inactivation
186
lyonization
inactivation random at early point in development
once inactivated, all progeny cells have same X-chromosome inactivated
187
anhidrotic ectodermal dysplasia
x-linked mutation causing sweat gland absence
males have no sweat glands
females have mosaic of D/d sections over body
188
X chromosome inactivation mechanisms
initiated from XIC (X inactivation centre)
produces 2 non-coding RNA transcripts (Xist/ Tsix)
leads to packaging of 1 X chr into v dense, compacted form of chromatin
189
polycistronic
mRNA corresponding to multiple genes whose expression is also controlled by single promoter/terminator
190
prokaryotic promoter
sigma factor recognises sequence upstream of gene, positioning RNA polymerase
191
holoenzyme
sigma factor + RNA polymerase
192
no. of RNA polymerases in prokaryotes
1
193
no. of RNA polymerases in eukaryotes
3
RNA polym 1, 2, 3
194
RNA polym 1
ribosomal RNA
195
RNA polym 2
all protein-coding genes
196
RNA polym 3
non-coding RNA's
197
RNA polym 2 promoters in eukaryotes
TATA box bound by transcription factor IID complex, recruiting RNA polym 2 and txn factors
5' cap added while RNA transcribed
198
transcription termination
transcripts end 10-35nt downstream of signal AAUAAA
RNA cut via endonuclease, releasing DNA
poly(A) tail added
199
prokaryotic gene expression alteration ways
alternative sigma factors (different -35/-10 sequences)
200
how many sigma factors do E coli have
7
201
mutation of sigma factor
affects expression of set of gene regulating
202
eukaryotic various transcription factors
allows stimuli to affect each promoter
203
microarray
DNA corresponding to each gene in sequence dotted onto slide
204
mutation in intron
doesn't affect coding sequence
could affect splicing
205
mutation in exon
alters coding sequence
206
intron/ exon boundaries function
define limits
recruit machinery removing introns from RNA ("spliceosome")
207
capping
1st modification made to RNA polym 2- transcribed RNA
208
polyadenylation
addition of poly(A) tail to RNA transcript
209
small ribosomal sub-unit
reading of mRNA
210
large ribosomal sub-unit
houses synthetic petidyl transferase centre
211
ribosome function
finds ORF start and interprets codons so as to pair w tRNA
212
how does start codon recognition differ in pro/eu?
eukaryotic mRNA has a cap
213
Shine-dalgarno sequence
large sub-unit binds to small sub-unit, translates/ initiates on downstream AUG
214
continuous variation
e.g. height/ foot size
polygenic
215
Mendel's first law
2 copies of each gene segregate in meiosis
216
Mendel's second law
copies of each gene segregate independent of other genes in Meiosis
217
when do Mendel's laws not apply
when both alleles are found on same chromosome
218
unlinked genes
meiosis independent assortment
219
linked genes
can't assort independently
220
Hardy-weinberg formulae
p^2 + 2pq +q^2= 1
221
Hardy Weinberg assumptions
infinitely large population
random mating
no evolutionary forces acting
222
when does the Hardy-weinberg principle not work?
gene flow
genetic drift
non-random mating
natural selection
223
examples of autosomal recessive conditions
albinism
cystic fibrosis
phenylketoneuria
sickle cell
haemochromatosis
tay-sachs
224
phenylketoneuria
inability to metabolize phenylalanine
225
Tay-sachs
neurodegeneration due to lack of N-acetyl-hexosaminidase
226
cystic fibrosis
thick mucus build-up due to CFTR absence
227
haemochromatosis
excess iron accumulation due to mutation in HFE gene of which there are 2 mutant alleles
228
HFE gene
regulates hepcidin production
229
hepcidin
iron regulatory hormone determining iron absorption from food/store release
230
examples of autosomal dominant conditions
huntington's
FASP
polycystic kidney disease
polydacyly
achondroplastic dwarfism
hypercholesterolaemia
231
achondroplastic dwarfism
FGFR3 mutation
failure to convert cartilage to bone
232
autosomal recessive pedigree
trait appears in progeny
horizontal pedigree pattern
233
autosomal dominant pedigree
trait appears in every generation
vertical pediree pattern
234
X-linked recessive disorders
haemophilia
muscular dystrophy
235
haemophilia
absence of factor VIII
blood doesnt clot
236
X-linked dominant disorders
hypophosphataemia
237
hypophosphatemia
vit-D resistant rickets
238
Leri-weill dyschondrosteosis
bone growth disorder
lack of SHOX gene copy
239
co-dominance
traits show up equally in F1
240
3 blood group alleles
I^a/I^b/i
241
incomplete dominance
F1 resembles neither parent
242
pleiotrophy
genes/alleles affecting more than one unrelated characteristic
e.g. manx phenotype
243
epistasis
gene modifying/masking phenotype of another gene
244
hypostatic
gene/allele being masked/modified
245
penetrance
number of members in population of specific genotype displaying the expected phenotype
246
expressivity
range of signs/symptoms that can occur in different people w genetic condition
247
polydactyly
incomplete penetrance as variable digits of progeny
248
complementation group
set of mutations mapping to same chromosomal locus, failing to complement when crossed
249
how is complementation skewed?
intragenic complementation
250
intragenic complementation
proteins w multiple functions forming multimers
251
linkage
genes on same chromosome, co-segregating in crosses
252
test-cross for 2 traits/genes
1. construct double heterozygote
2. cross w tester homozygous recessive for both traits
253
recombination frequency
(no. recombinants/ total progeny) *100
254
distance between 2 loci
1 centimorgan = 1 *10^6 Bp
255
syntenic genes
grouped in same way on chromosomes of 2+ species
256
synteny blocks
regions containing homologous genes
257
translocation
where 2 non-homologous chromosomes break and exchange fragments
258
acute myelogenous leukaemia
coding region for C-terminus of AML transcription replacement
required for haematopoiesis, coding region unrelated to other allele
259
centric fusion
2 telocentric chr fuse / generate new chromosome
260
robertsonian translocation
2 acrocentric p lost and 2q remaining fuse
261
result of robertsonian translocation
down syndrome/ patau syndrome
262
inversion
segment of chromosome becomes inverted with original position
263
2 types of inversion
pericentric
paracetric
264
paracentric
1 arm
265
pericentric
2 arms
266
result of chromosome inversions
small gene duplications/ large gene duplications prior to deletion of superfluous genes/ divergence of retained homologues
267
unequal crossover
strandbreak resulting in unequal crossover/ different no. repeat units
268
sister chromatid exchange
strandbreak on sister chromatids produces different repeat numbers
269
transcription factors in eukarya
bind motifs in promoter, promoting/inhibiting RNA pol II ability to initiate transcription
270
intron/ exon boundaries function
defines limits
recruit machinery removing introns from RNA
271
purpose of alternative splicing
can yield multiple products from each gene
272
bacterial translation
shine-dalgarno sequence recognised by small ribosomal subunit RNA via base-pairing
large binds to small sub-unit and translates downstream of AUG
small sub-unit binds CAP and large binds to first AUG
273
shine dalgarno function
allows independent translation of each ORF in polycistronic RNA
274
shine-dalgarno function
allows independent translation of each ORF in polycistronic mRNA
275
mutation in shine dalgarno
reduces specific ORF translation but not others
276
difference between pro/eu translation
pro is coupled / trans affects txn
eu is uncoupled/ trans doesn't affect txn
277
haplotype
association of number of polymorphic markers
278
variable number tandem repeats cause
unequal crossover/ replication errors
279
microsatellites
GC-rich, variant repeats of ~5 nucleotides
e.g. SSR/ STR
280
Paternity testing
PCR testing of minisatellites
DNA-17 profiling
281
DNA-17 profiling
detects STR'S/ STR loci
282
SNP/ single nucleotide polymorphism
single base differences
produce new sites for restriction endonucleases
283
RFLP
Restriction fragment length polymorphism
284
CpG islands
associated w promoters
enriched w CpG sites
identified when sequence unknown due to restriction enzyme clustering
associated with 5'
285
SNP map functions
locating genes associated w phenotypes
diagnosis of problems/ phenotypes
286
international HAPMAP project
SNP maps of human genome
haplotypes used as tool for genotyping and examination
analysed via bead/gene chips
287
bead chips
PCR amplification of whole genome
fragments DNA into smaller pieces and hybridizes primers bound to beads on chip
288
genome wide association study pros
informs patient care/screening/ human history
insurance
gene identification influencing phenotypes
288
multigene family
group of genes descending from common ancestor and therefore having similar functions / genotypes
289
how do multigene families arise
duplication events followed by mutation generating alterations in function/expression
290
function of hox genes
encode transcription factors influencing expression patterns of genes during development
291
homeobox
180 nucleotide segment wncoding DNA binding 'homeodomain'
292
primitive globin
monomeric
single O2 binding
293
thalassemia
alpha/beta chain production in unequal amounts leading to abnormal Hb
294
cause of thalassemia
mutation in a/b globin genes
severity depending on no. affected genes and mutation severity
295
B thalassemia
2b genes
1 mutated=minor
2 mutated= major
296
de novo generation
neutral/advantageous transcription of part of genome, evolving over time as new gene
297
In which cell-type is Baculovirus used as a cloning and expression vector for recombinant genes?
insect
298
Which enzyme is used to make a cDNA copy of mRNA?
reverse transcriptase
299
multiple cloning site
polylinker
