Recombinant Ppt

7/29/2019 Recombinant Ppt

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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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