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Recombinant DNA Technology Based On: Digest & Ligation

Recombinant DNA technology uses restriction enzymes to cut DNA at specific sites. Restriction enzymes cut DNA in different ways - Type I cuts nonspecifically, Type II cuts specifically producing sticky ends, and Type III cuts large fragments specifically. EcoRI is an example of a Type II restriction enzyme that produces sticky ends. Restriction enzymes are named based on the organism they are isolated from. The cut DNA can then be ligated back together to recombine genes in new ways. This technology has applications in producing proteins, developing gene therapies, and genetically modifying organisms.

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rengachen
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49 views

Recombinant DNA Technology Based On: Digest & Ligation

Recombinant DNA technology uses restriction enzymes to cut DNA at specific sites. Restriction enzymes cut DNA in different ways - Type I cuts nonspecifically, Type II cuts specifically producing sticky ends, and Type III cuts large fragments specifically. EcoRI is an example of a Type II restriction enzyme that produces sticky ends. Restriction enzymes are named based on the organism they are isolated from. The cut DNA can then be ligated back together to recombine genes in new ways. This technology has applications in producing proteins, developing gene therapies, and genetically modifying organisms.

Uploaded by

rengachen
Copyright
© Attribution Non-Commercial (BY-NC)
Available Formats
Download as PPT, PDF, TXT or read online on Scribd
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Recombinant DNA Technology

based on
Digest & Ligation
Restriction Endonuclease/enzyme

• H.O. Smith, K.W. Wilcox, &


T.J. Kelley (1968)
• Haemophilus influenzae
(HindI)
• Protective mechanism (R-M)
• Methylase – protects genome
• Endonuclease – against
phage
Types of restriction enzymes

• Type I (R-M): nonspecific cuts


• Type II (dimers): specific cuts
• Type III (R-M): large fragments, specific cuts
EcoRI (Palindromic – dimer)
Naming of Restriction Enzyme
•E Escherichia (genus)
•co coli (species)
•R RY13 (strain)
•I First enzyme identified from this organism

EcoRI Escherishia coli 5'GAATTC 5'---G AATTC---3'


3'CTTAAG 3'---CTTAA G---5'
BamHI Bacillus 5'GGATCC 5'---G GATCC---3'
amyloliquefaciens 3'CCTAGG 3'---CCTAG G---5'
AluI* Arthrobacter 5'AGCT 5'---AG CT---3'
luteus 3'TCGA 3'---TC GA---5'
TaqI Thermus aquaticus 5'TCGA 5'---T CGA---3‘
3'AGCT 3'---AGC T---5'
Plasmid Restriction Site Map
Digest Reaction Table

Single Digest Double Digest


Reaction Reaction
Plasmid (DNA):
0.5 – 1 ug
5 ul 5 ul
Enzyme 1
___EcoR 1______ 2.5 ul 1.2 ul
Enzyme 2
___BamH 1_____ None 1.3 ul
Buffer # _2_
5 ul 5 ul
BSA
1 ul 1 ul
Sterile H2O
36.5 ul 46.5 ul
Total volume 50 ul 60 ul
Ligation Reaction
5’ 3’
5’ 3’ GAT C C T A C T C AA C
C A T A G C AA G
G A T G A GT T G
G T A T C G T T C C T AG 3’ 5’
3’ 5’

5’ 3’
C A T A G C AA G GAT C C T A C T C AA C
Fix hydrogen bonds
G T A T C G T T C C T AGG A T G A G T T G
3’ 5’

5’ 3’
C A T A G C AA G GAT C C T A C T C AA C
Ligase fix phosphodiester
G T A T C G T T C C T AGG A T G A G T T G bonds (backbones)
3’ 5’

5’ 3’
C A T A G C AA G GAT C C T A C T C AA C
Ligation completed
G T A T C G T T C C T AGG A T G A G T T G
3’ 5’
Ligation Reaction Table

V:I (1:1) V:I (1:3)


Vector DNA
(1ng)
Insert DNA

Ligase (5%)

Ligase Buffer
(1/10)
Sterile H2O

Total Volume
Application of Recombinant
DNA Technology
• Efficient synthesis of
important proteins
• Medical therapeutic
molecule synthesis
• Possible genetic
disorder therapy
• Genetic modification of
organisms with high
economical values
• Create new DNA/RNA
sequences as research
tool

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