:Read more .

http://www.ykuwait.net/vb/showthread.php?t=22701#ixzz2O7YkmAc6

0971 ( )
.
( ) .
( )
. ( )
. .


.

.

.

.

.
. 6
:
-0 ) Cleavage of DNA(
( ) Restriction Nucleases
.
-2 (.) Gel Electrophoresis
-3 ) DNA sequencing(
.
.
-4 ) Nucleic acid hybridization(

.
-5 ) DNA cloning(
.
-6 ) DNA engineering(
.
DNA
:

.

 .

.0971
( ) Restriction Nucleases
.
() Nucleases Restriction
!
.
() .
.)Nucleases Restriction(
( ) .

.

)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

amyloliquefaciens H GGATCC BamHI Bacillus
EcoRI Escherichia coli RY13 GAATTC
Haemophilus aegyptius GGCC HaeIII
HindIII Haemophilus influenzae Rd AAGCTT
Haemophilus parainfluenzae GTTAAC HpaI
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
(
) .

() RFLP .Restriction Fragment Length polymorphism
RFLP's Map
.
.
.

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

()

.
.



.
. .

.
. ( ) Agarose gel
( .) gel Polyacrylamide

 511 (
..)A . 311
01111 ( ..

.


.



.
( Pulsed-field gel
.) electrophoresis

221111 2.5 ( .)C

(
) Ethidium Bromide .


.
) DNA sequencing(


.


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

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



.
:
-0 :
- ()specific primer
( ) .
- ( dGTP, dATP,
..)dCTP, and dTTP
- ( )DNA polymerase
- .
.
. .

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

.

( ) photomultiplier

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

.
( )Vectors
''
.
.

.
.
.

 )E.Coli( ( . )Yeast

.
.
) DNA( .) RNA(

.


.
(
)
.

selectable marker +cloning sites = YIP, yeast integrating plasmid

origin of replication YRP, yeast replicating plasmid = YIP + ARS
of YEP, yeast expression plasmid = YIP + 2 micron origin
replication
YCP, yeast centromere plasmid = YRP + centromere sequence
( : )Viral Vectors
( .)Phage
.
( )lambda phage .E. coli
.
. ( ) Cosmids
( cohesive sequence
)Cos sequence ( ) Phage ( ) Plasmid
41 ( ) kb41 0
( ) / P1-derived Artificial Chromosomes PAC 0
( ) Bacteriophage P1 ..
Plasmid Cosmid + Cos sequence from Lambda phage
P1 Phage + Plasmid PAC
plasmid-factor BAC Modified E.coli fertility
(:) Artificial Chromosomes

.
.
) Yeast Artificial Chromosomes / YAC(
511 ( .) kb 511

 )Telomeres 2( )Centromere(
(.)Autonomous replicating sequence ARS
) Artificial Chromosomes / BAC Bacterial(
051 ( ) kb 051
.))plasmid-factor E.coli fertility(
Vector
Standard plasmid 0- 10 KB
Bacteriophage 0-23 Kb Lambda
Cosmid 30-44 Kb
Bacteriophage P1 70-100 Kb 0
0 PAC 130- Kb 150 P1 P1Artificial chromosome
BAC Bacterial Artificial Chromosome
Kb 311
Mb 2-1.2 Yeast Artificial Chromosome YAC
(:)Cloning
.

. .

. )Cloning (
(.)Clone
... () Nature 385, 810-13, 1997
' ' ( ) 0997


.

.

.
3 :
-0
. ) Recombinant DNA technology (
-2 Reproductive cloning
-3 Therapeutic cloning
.

() ( .)
.

.
)Beta-Globin Gene (
%1.11115 ( 3 ).
) Dystrophin Gene (

 2.5 ( ) Megabases2.5 %1.18

.
. :
-0 ()Cell-Based DNA cloning
-2 ( )Cell-Free DNA cloning
( .)reaction PCR Polymerase chain
.
()Cell-Based DNA cloning
:
-0 ( )Vector
. (.)Restriction enzymes
DNA molecules by in Vitro Construction of recombinant -0
.)attachment to Replicon(Vector
-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
-0

.

( ) Vectors ( ).

. :
-0 ( )
.
-2 .
-3 .
.
.
( ) Ligase

 () Covalent Bond
.
.
.
-2
)E.Coli(
( 21)
.


.
.
-3

.


.

.
-4 .

.
.

.
)cDNA(
.