recombinant ppt
TRANSCRIPT
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Restriction
Enzymes
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INTRODUCTION
The major tools for the Rdnatechnology are the enzymes used forrecombinant DNA technology have
also been overproduced bygenetically engineered bacteria andtheir use is still increasing.
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Restriction Enzymes scan the DNA
codeFind a very specific set of
nucleotides
Make a specific cut
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Restriction Enzymes: Molecular
Scissors
Restrictionenzymes(endonuleases) cutDNA at specific
sequences What kinds of
bonds are brokenwhen restriction
enzymes cut? Covalent bonds (within a
single strand)
Hydrogen bonds (betweenstrands) as a result of the
strands coming apart
Hydrogen
bondCovalent bond
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Origins of Restriction
EnzymesNaturally found in different types
of bacteria
Bacteria use restriction enzymes to
protect themselves from foreignDNA
Bacteria have mechanisms to
protect themselves from theactions of their own restrictionenzymes
Have been isolated and sold for
use in lab work
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Examples of Restriction
EnzymesEnzyme Organism source Recognized
Sequence
EcoRI Escherichia coli 5' GAATTC 33' CTTAAG5
TaqI Thermus aquaticus 5' TCGA 33' AGCT5
HindIII Haemophilus influenzae 5'AAGCTT 33'TTCGAA5
BamHI Bacillusamyloliquefaciens
5' GGATCC 33' CCTAGG5
AluI Arthrobacter luteus 5' AGCT 33' TCGA 5
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Sticky Ends vs. Blunt Ends
When restriction enzymes cut,they produce either
Sticky ends (single stranded sections
at the ends)Blunt ends 5 - - - G A A T T C - - - 3
I I I I I I I
3 - - - C T T A A G - - - 5
Sticky ends
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blunt end
sticky end
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HaeIII
HaeIII is a restriction enzyme thatsearches the DNA molecule untilit finds this sequence of four
nitrogen bases.
5 TGACGGGTTCGAGGCCAG 33 ACTGCCCAAGGTCCGGTC 5
5 TGACGGGTTCGAGGCCAG 3
3 ACTGCCCAAGGTCCGGTC 5
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Once the recognition site was found
HaeIII could go to work cutting
(cleaving) the DNA
5 TGACGGGTTCGAGGCCAG 3
3 ACTGCCCAAGGTCCGGTC 5
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These cuts produce what scientists call
blunt ends
5 TGACGGGTTCGAGG CCAG 3
3 ACTGCCCAAGGTCC GGTC 5
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blunt ends and sticky endsRemember how HaeIII produced a
blunt end?
EcoRI, for instance, makes astaggered cut and produces a sticky
end5 GAATTC 3
3 CTTAAG 5
5 GAATTC 33 CTTAAG 5
5G AATTC 3
3 CTTAA G 5
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Some more examples of restriction sites of
restriction enzymes with their cut sites:
HindIII: 5 AAGCTT 3
3 TTCGAA 5
BamHI: 5 GGATCC 3
3 CCTAGG 5
AluI: 5 AGCT 3
3 TCGA 5
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ADVANTAGES
First, they are significantly lessexpensive.
Second, purification of enzymes iseasier and more rapid, resulting inmore homogeneous enzymes withhigher specific activities.
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DNA LIGASES
DNA Ligases close nicks in thephosphodiester backbone of DNA.
Biologically, DNA Ligases are essentialfor the joining of Okazaki fragmentsduring replication, and for complitingshort-patch DNA synthesis occuring
in DNA repair process.
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DNA POLYMERASE
A DNA polymerase is an enzyme thatcatalyzes the polymerization ofdeoxyribonucleotides into a DNA strand.
DNA polymerases are best known for their
role in DNA replication in which thepolymerase" reads an intact DNA strandas a template and uses it to synthesizethe new strand.the process copies apieces of DNA.
DNA polymerases use a mg++ forcatalytic activitiy.
Type . Pol I, pol II, pol III
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EXONUCLEASE
Exonucleases are enzymes that work bycleaving nucleotides one at a time fromthe end of a polynucleotide chain.
A hydrolyzing reaction that breaks
phosphodiester bonds at either the 3 orthe 5ends occurs.
Eukaryotes and Prokaryotes have 3 typesof exonucleases involved in the normal
turnover of mRNA :3, to 5 exonuclease,which is a dependent decapping protein,5to 3 exonuclease, an independent protein,and poly(A)- specific 3 to 5 exonuclease.
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TERMINAL DEOXYNUCLEOTIDYL
TRANSFERASE Terminal Deoxynucleotidyl Transferase,
also known as TdT and terminaltransferase.
TdT catalyses the addition of nucleotides
to the 3 terminus of a DNA molecule. The preferred substrate of this enzyme is
a 3 overlapping, but it can also addnucleotides to blunt or recessed 3 ends.
Cobalt is a necessary cofactor, howeverthe enzymes catalizes reaction upon Mgand Mn administration in vitro.
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ALKALINE PHOSPHATASE
Alkaline phosphatase(ALP), is ahydrolase enzyme responsible forremoving phosphate groups from
many types of molecules, includingnucleotides, proteins and alkaloids.
The process of removing the
phosphate group is calledDephosphorylation.
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POLYNUCLEOTIDE KINASE
Polynucleotide kinase (PNK) is anenzyme that catalyzes the transfer ofa phosphate from ATP to the 5 end
of either DNA or RNA.
The enzymatic activity of PNK isutilized in two types of reactions:
Forward reaction.
Exchange reaction.
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REVERSE TRANSCRIPTASE
This enzyme by using the templateof RNA ,synthesize the new strand ofDNA .
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