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 ))

485 () . .

() ֡ . .
. . . .
. (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 (40kb ) 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 (500 kb ). (2 Telomeres) (Centromere) (Autonomous replicating sequence ARS). (Bacterial Artificial Chromosomes / BAC ) 150 (150 kb ) (E.coli fertility plasmid-factor)).
Vector
Standard plasmid 0- 10 KB
Lambda Bacteriophage 0-23 Kb
Cosmid 30-44 Kb
1 Bacteriophage P1 70-100 Kb
1 P1 P1Artificial chromosome PAC 130- Kb 150
Bacterial Artificial Chromosome BAC 300 Kb
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 (2.5Megabases ) 0.08% . . :
1- (Cell-Based DNA cloning)
2- (Cell-Free DNA cloning) ( Polymerase chain reaction PCR). .

(Cell-Based DNA cloning)
:

1- (Vector) . (Restriction enzymes).

1- Construction of recombinant DNA molecules by in Vitro attachment to Replicon(Vector).

2- (Bacteria ) (E,coli ( Yeast).

2-Transformation using bacteria or yeast

3- . (Culture Plates) .

3- Selective propagation of cell clones.

4- .

3- Isolation of recombinant DNA clones
1-
. ( Vectors ) ( ). . :
1- ( ) .
2- .
3- . . . (Ligase ) (Covalent Bond )
. . .
2-
(E.Coli) ǡ ( 20 ) . . .
3-
. ɡ . .
4- .
. . . (cDNA) .