Topics in Nanobt Lecture 6 2006 2007

8/7/2019 Topics in Nanobt Lecture 6 2006 2007

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TOPICS IN (NANO)BIOTECHNOLOGY

Lecture 6

30th October, 2006

PhD Course


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Overview

So we have looked at what is DNA and whatis a gene.

We also looked at DNA replication andprotein synthesis, and the path from the geneto protein

We know what proteins are and especiallyabout two specific sets of proteins enzymesand antibodies

This week we will look at Recombinant DNAtechnology


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Production of a unique DNA molecule byjoining together two or more DNA fragmentsnot normally associated with each other

DNA fragments are usually derived fromdifferent biological sources

D efinition of recombinant DNA


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Enzymes for Manipulating DNA.

Vectors for gene cloning.

Cloning of rDNA.

Characterization of Cloned Genes

Construction of Genomic libraries.

Applications of the rDNA technology

D iscussion of r DNA technology


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History of Recombinant DNA technology

Antibiotics such as penicillin, thesulfonamides and streptomycin gave muchhope

However, in the 50s theye starting to fightback, becoming increasingly resistant toantibiotics

In just a few years 60-80% of bacteria showedresistance not just to one drug, but to multipledrugs

The genes responsible for infectious drugresistance were plasmids, genetic elementsthat could replicate themselves independently.

In different plasmids, the replication regionencodes traits not essential to the bacterialhost.

Antiobiotic resistance is one of these traits.


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History of Recombinant DNA technology

In the late 60s, it was shown that CaCl 2 made the cells of E.coli permeable so that they could take up DNA, butcould not grow E.coli cells with genetic property changes.

In 1971 Cohen, exploited the antibioticresistance of the plasmids toselectively enrich offspring thatcontained cell propogating plasmids.

In late 1972, Berg reported onmethods for joining fragments of DNAoutside of cells using ligases.

Endonucleases, or restrictionsenzymes, would however, provide thetool for linking DNA.


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U na cerveza y ...In Nov. 1972, Berg,Boyer and Cohen metup at a deli bar inHonololu, anddiscussed the

endonuclease thatBoyer was working on,and that night they

dreamed of the collaborative project that would be the truestart of recombinant DNA technology. In March 1973, the

pair produced DNA fragments using Boyers technique andjoined them to plasmids using Bergs technique, and thenintroduced them into bacteria using Cohens technique.

The first demonstration of DNA cloning had been achieved.


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But lets look at it in moredetail....


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Recombinant DNA technology


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R ecombinant DNA technology

The two essential elements of recombinantDNA technology are:

1. Restriction endonucleases2. Vectors for gene cloning


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Restriction endonucleases


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W hat is a restriction enzyme? There are two classes of restriction enzymes:

Type I Cuts DNA on both strands but at non-

specific location Random imprecise cuts Not very useful for rDNA applications

Type II Cuts both strands of DNA within theparticular sequence recognised by therestriction enzyme


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W hat is a restriction enzyme?

Restriction enzymes (or endonucleases) arebacterial enzymes that cut DNA at veryspecific sequences

They generally cut in a staggered manner,leaving sticky ends but some enzymesgenerate blunt ends (i.e. Cut DNA in themiddle)

Their biological function is to destroy invadingforeign DNA


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Each bacteria has different restrictionenzymes

Enzymes from E.coli cells cut GAATTC/CTTAAG

Enzymes from B. Amyloloquefaciens cutGGATCC/CCTAGG

The restriction enzymes are named after the

organism from which they were derived

EcoRI from E.coli BamHI from B. Amyloloquefaciens


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Restriction enzymes are used to makerecombinant DNA and gene cloning andgenetic engineering were made possible bythese enzymes

Over 200 different restriction enzymes arecommercially available (some are VERYexpensive)

DNA ligase sticks the ends back together


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Recombinant DNA technology can be used toisolate a genomic clone from DNA or for theisolation of human cDNA

Isolating a genomic clone provides a piece of DNA identical in base sequence to thecorresponding stretch of DNA in the cell andis often designed to contain a specific gene

Isolating human cDNA is used for geneexpression. Human cDNA(c=complementary) is double stranded DNA

copy of mRNA but WITHOUT introns


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V ectors for gene cloning


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V ector requirements

Dependent on design of experimental system Most vectors contain a prokaryotic origin of replication

Can replicate along with the host cell Autonomously

By integration in the chromosome

Antibiotic resistance genes and/or other selectable markers

Contain one or more unique sites for insertion

of foreign DNA


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V ector typesPlasmids (upto about 20kb insert)Bacteriophage

P vectorsCan insert fragments of DNA up to 25 kb.Can introduce into cells at a very high efficiency(10-100kb depending on the type ofbacteriopage)

Cosmid (35-45kb)

Combination of bacteriophage and plasmid

BAC vectors (bacterial artificial chromosomes )

Contain sequences from the E. coli F plasmid present at one copy per cell. Can clone up to 200 kb

per BAC clone


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W hat is a plasmid? Plasmids are small, extrachromosomal piecesof bacterial DNA that are often antibioticresistant

They are shuttle vectorsto create, produce, and maintainrecombinant DNA

An example of one of the firstplasmids is pBR322

Both Amp & Tet resistant, Several unique restrictionsites

pUC18 now the most commonly used Derivative of pBR322

Smaller, Higher copy number per cell, Multiple

cloning sites


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W hat is a plasmid?


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lacZ gene Gene encoding for enzyme F-galactosidase

Polylinker resides in the middle

Enzyme activity can be measured as marker of gene insertion Disrupted gene nonfunctional WHITEWHITE Intact gene functional BLUE

Amp resistance gene still present, Tetresisitance gene omitted


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W hat is a bacteriophage?


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L ambda vector

Bacteriophage lambda ( P) infects E.coli

Double stranded linear DNA vector, suitable for

library construction

Can accomodate large segments of foreignDNA, central 1/3 is a stuffer fragment

Can be substituted with any DNA fragment of similar size Can accomodate } 15kbp of foreign DNA


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L ambda vector


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V ectors for eukaryotic cells

Shuttle vectors- Hybrid molecules designed for use in multiple cell types- Multiple ORIs allow replication in both prokaryotic and eukaryotic

host cells allowing transfer between different cell types

YAC vectors (Yeast artificial chromosomes)- Contains sequences required to replicate and maintainchromosome in budding- yeast (like P, end up as a linear molecules)

- a yeast origin of replication, a centromere, and a telomere at eachend.

- Can clone >2,000 kb (2 Mb).

- Agrobacterium TumefaciensPlants

- BaculovirusInsect cells


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C omparison of different vectors


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Getting Recombinant DNA into cells


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Procedure for cloning DNA in a plasmidvector


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Only bacteria which havetaken up plasmid grow onampicillin.

Blue-white selection: white colonies have insert blue colonies have no

insert

To see blue color, add IPTG(an inducer of F-galactosidaseexpression) and Xgalsubstrate.

I nsertion into a Plasmid can be D etected byD isruption of F-gal


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