1970 ( )

. ( )
. (
) . ( )
. .


.
.

.

.
.
. 6
:
-1 ) Cleavage of DNA( ) Restriction Nucleases(
.
-2 (.) Gel Electrophoresis
-3 ) DNA sequencing( .
.
-4 ) Nucleic acid hybridization(
.
-5 ) DNA cloning( .
-6 ) DNA engineering(
.
DNA :

.
.
.1970
( ) Restriction Nucleases
.
() Restriction Nucleases
!
.
() . .)Restriction Nucleases(
( ) .

.
)Methyl (
) Methylation at an A or a C residue(
.
. .
.
)Blunt ends( ( ) ,Staggered cuts
'' .

.)Recombinant DNA(


.
. .
() Hpa I 6 ( )GTTAAC
( ) Eco RI 6 ( .)GAATTC

 ( ) Hemophilus parainfluenzae
.
( ) Escherichia coli .
Recognition Sites

BamHI Bacillus amyloliquefaciens H GGATCC
EcoRI Escherichia coli RY13 GAATTC
HaeIII Haemophilus aegyptius GGCC
HindIII Haemophilus influenzae Rd AAGCTT
HpaI Haemophilus parainfluenzae GTTAAC
HpaII Haemophilus parainfluenzae CCGG
MboI Moraxella bovis GATC
NotI Nocardia otitidis-caviarum GCGGCCGC
SfiI Streptomyces fimbriatus GGCCNNNNNGGCC
TaqI Thermus aquaticus TCGA
(:) Restriction fragments

. .
. .
() .2
(
( .)Gel Electrophoresis
( )
.
() Restriction Fragment Length polymorphism. RFLP
RFLP's Map
.
.
.

.
.
)) BamHI, EcoRI, and HindIII
48 5 ()
.
.

()
.
.



.
. .
.
. ( ) Agarose gel
( .) Polyacrylamide gel
500
( ..)A .
300 10000 ( ..

.



.


.
( .) Pulsed-field gel electrophoresis

220000 2.5 ( .)C

) Ethidium Bromide (
.

.
) DNA sequencing(


.

. .
( ) Enzymatic method ( .) Chemical
.
(:) Enzymatic method
() Sanger procedure .
. .)dideoxy sequencing(

()
.
( ) deoxynucleotides (dNTPs
)OH (

. .
()OH ) H( ( )ddNTPs ( ) dNTPs

.
:
-1 :
- ()specific primer
( ) .
- (..)dGTP, dATP, dCTP, and dTTP
- ( )DNA polymerase
- .
. .
.

.
-2
.
-3 ( )Autoradiography .
-4 .
.

( ) photomultiplier
.
( ) fluorescent
( )
.
.
( %99)
.

 ( )Vectors
''
.
.

.
.
. )E.Coli(
( . )Yeast
. .
) DNA(
.) RNA(
.

.

( )
.

YIP, yeast integrating plasmid = selectable marker +cloning sites

YRP, yeast replicating plasmid = YIP + ARS origin of replication
YEP, yeast expression plasmid = YIP + 2 micron origin of replication
YCP, yeast centromere plasmid = YRP + centromere sequence
( : )Viral Vectors
( .)Phage
. ( )lambda phage
.E. coli .
. ( ) Cosmids
( cohesive sequence )Cos sequence
( ) Phage ( ) Plasmid 40 ( ) kb40
1 ) / PAC P1-derived Artificial Chromosomes( 1
( ) P1 Bacteriophage ..
Cos sequence from Lambda phage + Plasmid Cosmid
P1 Phage + Plasmid PAC
Modified E.coli fertility plasmid-factor BAC
(:) Artificial Chromosomes

.
. Yeast Artificial Chromosomes / (
) YAC 500 ( .) kb 500
)Telomeres 2( )Centromere( ( Autonomous
.)replicating sequence ARS ) Bacterial Artificial Chromosomes / BAC(
150 ( ) kb 150 E.coli fertility (
.))plasmid-factor
Vector
Standard plasmid 0- 10 KB
Lambda Bacteriophage 0-23 Kb
Cosmid 30-44 Kb
Bacteriophage P1 70-100 Kb 1
1 P1 P1Artificial chromosome PAC 130- Kb 150
Bacterial Artificial Chromosome BAC Kb 300
Yeast Artificial Chromosome YAC 0.2-2 Mb
(:)Cloning
.

 .
.
. (
)Cloning (.)Clone
... () Nature 385, 810-13, 1997 ' '
( ) 1997

.

.
.
3 :
-1 (
. ) Recombinant DNA technology
-2 Reproductive cloning
-3 Therapeutic cloning
.
()
( .)
.
.
)Beta-Globin Gene (
%0.00005 ( 3 ).
) Dystrophin Gene ( 2.5 ( ) Megabases2.5
% 0.08 .
.
:
-1 ()Cell-Based DNA cloning
-2 ( )Cell-Free DNA cloning ( Polymerase chain
.)reaction PCR .
()Cell-Based DNA cloning
:
-1 ( )Vector .
(.)Restriction enzymes
.)Construction of recombinant DNA molecules by in Vitro attachment to Replicon(Vector -1
-2 ( ) Bacteria
E,coli( ( .)Yeast
Transformation using bacteria or yeast-2
-3 .
( )Culture Plates .
.Selective propagation of cell clones -3
-4 .
Isolation of recombinant DNA clones -3
-1

.
( ) Vectors
( ) .
. :
-1 ( )
.
-2 .
-3 .
.
.

 ( ) Ligase ( Covalent
) Bond .
.
.
-2
)E.Coli(
( 20)
.

. .
-3

.


.
.
-4 .

. .

.
)cDNA(
.