chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es
DESCRIPTION
Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es. Synthesis of pyrimidine bases. Transforma tions of pyrimidine bases. Syntheses of puri ne bases. Transforma tions of puri ne bases. Synthesis of nu c leosid es. Synthesis of nu c leosid es. - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/1.jpg)
Chemical syntheses of nucleobases, nucleosides, nucleotides a oligonucleotides
N
N NH
N
NH2
N
N N
N
O
OH OH
HO
NH2
N
N N
N
O
OH OH
O
NH2
PO
HOOH
N
N N
N
O
OH OH
O
NH2
PO
OOH
POPHOOH
O
OH
O
N
NN
N
NH2
O
O
OP-OO-
O
N
NH2
ON
O
O
OP-O O
N
NH2
ON
O
OH
OP-O O
![Page 2: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/2.jpg)
Synthesis of pyrimidine bases
H2N
NH
SEtN
NH
O
SEt
R
R'
HCl, H2O
NH
NH
O
O
R
R'H2N
NH2
O
OEt
O
Br
R
R'
OEt
O
OMe
R
R'
OEt
O
O
R
R'+or or
heterocyclization
NH
N
NH2
O
R
R'H2N
NH2
O
CN
OMe
R
R'
CN
O
R
R'R'
CN
+or or
heterocyclization
![Page 3: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/3.jpg)
Transformations of pyrimidine bases
N
NH
O
OR
N
NH
N
N
N
OR
N
NH3
N
N
NH2
OR
N
NH
O
OR
I
I2, CAN
R-M (organometallic)
catalysis N
NH
O
OR
R
![Page 4: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/4.jpg)
Syntheses of purine bases
N
N NH
N
X
X'
N
N NH2
NH2
X
X'
HN
H2N NH
N
X
X"X'-COY X"-COY+ +heterocyclization heterocyclization
X"
OCN
OEtNH2 O
H2N NH
N
OEtHN
N NH
N
O1. NH3
H2N
HNH+ 2. HC(OEt)3
N
N N
N
NH2
R
N
N Cl
NO2
NH2
N
N NH
NO2
NH2
R
RNH2
2. HC(OEt)3
1. reduction
![Page 5: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/5.jpg)
Transformations of purine bases
N
N N
N
NH2
R
HNO2, AcOH
HN
N N
N
O
R
POCl3, DMFN
N N
N
Cl
R
N
N N
N
I
R
HI
i-AmONO, CH2I2
NH3
R'-M
catalysis
R'-M
catalysis
N
N N
N
R'
R
![Page 6: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/6.jpg)
O
OBz
BzO
OBz
N
N
OEt
O
O
OH
HO
OH
N
N
NH2
OCl
O
OBz
BzO
OBz
N
N
OEt
OEt
NaOH O
OH
HO
OH
N
NH
O
O
CH3CN, 0°C then reflux
NH3, MeOH
Quaternization method
Synthesis of nucleosides
O
OBz
BzO
OBz
N
NH
O
O
OAc
N
N
OTMS
OTMS
Silyl Base method
1. TMSTf (from -Cl) or SnCl4 (from -OAc)2. H2O
![Page 7: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/7.jpg)
O
OBz
BzO
R
N
N N
N
X
Y
N
N NH
N
Y
X
ClO
OBz
BzO
R
OAcO
OBz
BzO
RN
N NH
N
Y
X
O
OBz
BzO
R
N
N N
N
X
Y
O
OBz
BzO
R
N
N N
N
X
Y
O
OBz
BzO
R
N
N N
N
X
Y
SnCl4
CH3CN, reflux
base (NaH)
CH3CN, reflux
9--
7--
7--
9--
Synthesis of nucleosides
O
OBz
BzO
OO
Ph
O
OBz
BzO
OBz
NuNu
9--
Neighboring group participation
![Page 8: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/8.jpg)
N
NN
N
NH2
O
OHOH
OP-OO-
O
N/CH replacement
CH/N replacement; substitution
substitution
configuration; substitution
sulfa, aza, carba ... analogues 1
2345 67
8 9
• acyclic nucleoside/nucleotide analogues• cyclonucleosides• fused and bicyclic analogues• homonucleosides• modified oligomucleotides
Modifications of Nucleosides and Nucleotides
![Page 9: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/9.jpg)
HN
N N
N
OHO
O
Didanosine, ddI
N
OHO
HN
O
O
N3
Zidovudine, AZT
N
N N
N
HO
NH2
OH OHAristeromycin
HN
N N
N
OHO
O
H2N
Acyclovir
N
N N
N
NH2
O PO
OHOH
Adefovir
N
N N
N
OHO
NH2
OH
F
OH
Fludarabine
N
N
O
NH2
AraC
antiviral antineoplastic
N
N N
N
OHO
NH2
OH OH
R
adenosine receptorsantagonists -antihypertensive
OHO
OH
OH
Biological Activity of Nucleoside Analogues
![Page 10: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/10.jpg)
BO
OH
HO
R
POCl3
P(O)(OMe)3
H2O (1 eq.)
BO
OH
O
R
PClO
Cl
selective
H2O work-upB
O
OH
O
R
PHOO
HO
Synthesis of nucleotides
1. (NHBu3)2H2P2O7NBu3, DMF
2. TEAB, H2O
BO
OH
O
R
POO
-OPOPO
O O
O- O-
1. (PhO)2POCl or DCC,morpholine2. (NHBu3)2H2P2O7
Enzymatic: nucleoside kinase
![Page 11: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/11.jpg)
BO
OH
HO
R
BO
OH
O
R
PHOO
HB
O
OH
O
R
PHOO
HOH3PO3
SO2 NN
NN
I2, H2O, Py
use of P(III) reagents
Synthesis of nucleotides
![Page 12: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/12.jpg)
Synthesis of oligonucleotides
1. Phosphodiester method2. Phosphotriester method3. H-Phosphonate method4. Phosphoramidite method
N
NN
N
NH2
O
O
OP-OO-
O
N
NH2
ON
O
O
OP-O O
N
NH2
ON
O
OH
OP-O O
![Page 13: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/13.jpg)
BO
O
O
O-P-O O
PGB
O
O
O
BO
O
OP-O O
PG
PG
BO
O
HO
PG
DCC
N C NDCC
+days30-80%
Phosphodiester method
History…
![Page 14: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/14.jpg)
Phosphodiester method
BO
O
O
O-PO O
DMTr
NC
O
OCH3
BO
O
O
O-PO O
DMTr
N
BO
OH
ODMTr
OHNO
OCH3
OP
N
N
NNN
N
N
NN
OP
N
O
NNN
N
N
NO2
1.
2. HOCH2CH2CN3. H2O
1.
2.+ TPSCl, MeIm
3. aq. NaOH
![Page 15: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/15.jpg)
Phosphotriester method
BO
O
O
O-PO O
PG
NC
BO
O
HO
PG
BO
O
O
BO
O
OPO O
PG
PG
NC
SO
OCl
+
![Page 16: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/16.jpg)
Phosphotriester method
BO
O
O
O-PO O
PG
NO
OCH3
BO
O
HO
PG
BO
O
O
BO
O
OPO O
PG
PG
NO
OCH3
SO
OCl
+minutes80-99%
![Page 17: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/17.jpg)
Phosphotriester method
BO
O
O
BO
O
OPO O
PG
PG
NO
OCH3
thiophenol
BO
O
O
BO
O
OP-O O
PG
PG
![Page 18: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/18.jpg)
O
PO
ROO
O BOH
PO
OO
O OB
N
CH3O
PO
OO
O OB
N
CH3O
OSO2Ar
OP
OO
HO
O
ONO
OCH3
SO2R
R
R
![Page 19: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/19.jpg)
BO
O
O
O-PH O
PGBO
OH
OPG
OPO
O
Cl
TEABtriethylammonium bicarbonate
1. PCl3/imidazole/Et3N2. hydrolysis
H-Phosphonate method
![Page 20: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/20.jpg)
BO
O
O
O-PH O
PG
BO
O
HO
PG
BO
O
O
BO
O
OPH O
PG
PG
+seconds98-99.5%
O
Cl
H-Phosphonate method
![Page 21: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/21.jpg)
OCl OCl
O
O
O
O
PH O
OHO
![Page 22: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/22.jpg)
BO
O
O
BO
O
OP-O O
PG
PGB
O
O
O
BO
O
OPH O
PG
PG
I2
oxidation
H-Phosphonate method
nucleophilicsubstitution
Nu-
BO
O
O
BO
O
OPNu O
PG
PG
Nu = S, NR2, BH3
![Page 23: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/23.jpg)
BO
O
O
PN O
PG
CN
BO
OH
OPG
ClPN O
CN
EtN(i-Pr)2
NPN O
CN
+ tetrazol
Phosphoramidite method
![Page 24: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/24.jpg)
BO
O
O
PN O
PG
CNB
O
O
HO
PG
BO
O
O
P O
PG
BO
O
O
PG
CN
N
NN
HN
N
NN
HN
EtS N
N
H
OTf
+seconds99-99.8%
Phosphoramidite method
![Page 25: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/25.jpg)
O
O
P OCH2CH2CN(i-C3H7)2N
H
NN
NN
O
O
P OCH2CH2CN(i-C3H7)2N
NN
NN
H
O
O
P OCH2CH2CN
NN
NN
OCH2CH2CN
O
P
OO
OHO
![Page 26: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/26.jpg)
BO
O
O
P O
PG
BO
O
O
PG
CNO
BO
O
O
P O
PG
BO
O
O
PG
CN
BO
O
O
P OH
PG
BO
O
O
PG
O
NH3oxidation
I2, H2O, lutidine-elimination+deacetylation of bases
Phosphoramidite method
![Page 27: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/27.jpg)
N
NN
N
HN
O
N
N
HN
O
O
NH
N
O
O
NH
N
O
O
NH
NN
N
O
NH
O
ABz CBz
GiBu
unprotected
Protection of bases
![Page 28: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/28.jpg)
BO
OH R
HO
BO
OH R
PGO
O
BO
OH R
TrO
MMTr
O
OCH3
DMTr
BO
OH R
HO
O
OCH3
OCH3
5'
Tr
TrCl, PyH2, Pd/Cor H+
DMTr - TFA or TCA
Protection of sugar
![Page 29: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/29.jpg)
BO
OH OH
HO
BO
OH OH
DMTrO
BO
OH OPG
RO
Cl Si O Si Cl
TBDMS
OSi
BO
O OH
O
Si
SiO
BO
OH OTBDMS
DMTrO
TFA
Bu4N+ F-
(TBAF)
N
H3CO
F
O
N
OCH3
F
BO
OH OH
DMTrO
BO
O OFpmp
O
Si
SiO
2'
TBSMDCl, imidazole
Fpmp
Ribonucleotides
+ 3'-isomer
Protection of sugar
![Page 30: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/30.jpg)
Solid-phase oligodeoxyribonucleotides
H2N
HN O O Si CPG
O
OAc OMe
OMeB
O
O
DMTrO
O
O
O
NO2
NH
HN O O Si CPG
O
OAc OMe
OMe
BO
O
DMTrO
O
O
Attachment to solid support
CPG = controlled pore glass
10-50mol/g
![Page 31: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/31.jpg)
Solid-phase oligodeoxyribonucleotides
OB1
DMTrO
TCAO
B1
HO5'
3'5'
3'
OB2
DMTrOP
N
OCH2CH2CN
OB1
OOB2
DMTrOP
OCH2CH2CN
5'3'
5'3'
5'3'
tetrazole
OB1
OOB2
DMTrOP
OCH2CH2CN
O
I2, H2O, pyridine
5'3'
5'3'
TCAOB1
OOB2
HOP
OCH2CH2CN
O
5'3'
5'3'
next coupling
OB1
HOAc2OO
B1
AcO
5'3'
capping
5'3'
unreacted
![Page 32: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/32.jpg)
Solid-phase oligodeoxyribonucleotides
1. Detritylation2. Coupling with phosphoramidite3. Capping4. Oxidation5. Detritylation…..6. Deprotection and release (aq. NH3, 50°C, 5h)7. Purification (HPLC, GEP)
Total yield Yn= Yi(n-1) 20-mer 80% -> 1.4%
90% -> 13%99% -> 82%
99.8% -> 96%
![Page 33: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/33.jpg)
REGULATION OF GENE REGULATION OF GENE EXPRESSIONEXPRESSION
ANTISENSE STRATEGYANTISENSE STRATEGYinteraction with RNAinteraction with RNA
ANTIGENE STRATEGYANTIGENE STRATEGYinteraction with DNAinteraction with DNA
APTAMER STRATEGYAPTAMER STRATEGYinteraction with proteinsinteraction with proteins
![Page 34: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/34.jpg)
Hybrid duplexHybrid duplexm-RNA*DNA-oligomerm-RNA*DNA-oligomer
TranslationTranslationarestarest
No protein No protein synthesissynthesis
![Page 35: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/35.jpg)
Hybrid duplexHybrid duplexm-RNA*DNA-oligomerm-RNA*DNA-oligomer
DNA-oligomerDNA-oligomer
RNase HRNase H
Products of m-RNA cleavage
![Page 36: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/36.jpg)
O
O
O
PHO O
OO
O
B1
B2
Resistance against nucleaseResistance against nucleasecleavagecleavage
High affinity towards targetHigh affinity towards target sequences of RNA/DNAsequences of RNA/DNA
Selectivity – discrimination Selectivity – discrimination between DNA and RNA between DNA and RNA
Low non-specific binding andLow non-specific binding andhigh sensitivity to mismatch pairshigh sensitivity to mismatch pairs
REQUIREMENTS FOR MODIFIED REQUIREMENTS FOR MODIFIED OLIGONUCLEOTIDESOLIGONUCLEOTIDES
Activation of RNase H cleavageActivation of RNase H cleavageabilityability
![Page 37: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/37.jpg)
OPO
HS
O
O
B
BS
OPO
HO
O
O
B
BS
OCH2CH2OCH3
OPO
HO
O
O
B
B
OCH2CH2OCH3
S
O B
X
OP OO
HO
O B
X
OP X
HNHO
O
O
B
BOP OO
O
O
B
BH3B
N
OB
O
N
OB
P ONCH3
CH3OP OO
CH3
O
O
B
B
!! !!
!! !!!!
MOST IMPORTANT MODIFICATIONS MOST IMPORTANT MODIFICATIONS OF INTERNUCLEOTIDE LINKAGESOF INTERNUCLEOTIDE LINKAGES
![Page 38: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/38.jpg)
PPeptide eptide NNucleic ucleic AAcidscids
NN
NN
NN
.........N
O OO
B1
HH
OB2
H
O
H
OB3
O
........
![Page 39: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/39.jpg)
Classical synthesis of genes (duplexes DNA)
1. Synthesis of oligonucleotide fragments (20-40-mers, cohesive ends)
2. 5’-Phospohorylation (enzymatic or chemical)3. Ligation – T4 DNA ligase
![Page 40: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/40.jpg)
PCR (Polymerase Chain Reaction)
1. Add primers complementary to flanking sequence2. Add all nucleoside triphosphates and thermostable
DNA polymerase3. Heat 95°C 15s - strand separation4. Cool 54°C – hybridization5. Heat 72°C (optimal temp.) – DNA synthesis
![Page 41: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/41.jpg)
![Page 42: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/42.jpg)
![Page 43: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/43.jpg)
![Page 44: Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es](https://reader035.vdocument.in/reader035/viewer/2022081604/56815e17550346895dcc7575/html5/thumbnails/44.jpg)
DNA cloning