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PhD Course . TOPICS IN (NANO) BIOTECHNOLOGY Lecture 6. 30th October, 2006. Overview . So we have looked at what is DNA and what is a gene. We also looked at DNA replication and protein synthesis, and the path from the gene to protein

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PhD Course

TOPICS IN (NANO) BIOTECHNOLOGY

Lecture 6

30th October, 2006


Overview
Overview

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

  • We also looked at DNA replication and protein synthesis, and the path from the gene to protein

  • We know what proteins are – and especially about two specific sets of proteins – enzymes and antibodies

  • This week we will look at Recombinant DNA technology


Definition of recombinant DNA

  • Production of a unique DNA molecule by joining together two or more DNA fragments not normally associated with each other

  • DNA fragments are usually derived from different biological sources


Discussion of rDNA technology

  • Enzymes for Manipulating DNA.

  • Vectors for gene cloning.

  • Cloning of rDNA.

  • Characterization of Cloned Genes

  • Construction of Genomic libraries.

  • Applications of the rDNA technology


History of Recombinant DNA technology

  • Antibiotics such as penicillin, the sulfonamides and streptomycin gave much hope

  • However, in the 50s theye starting to fight back, becoming increasingly resistant to antibiotics

  • In just a few years 60-80% of bacteria showed resistance not just to one drug, but to multiple drugs

  • The genes responsible for infectious drug resistance were plasmids, genetic elements that could replicate themselves independently.

  • In different plasmids, the replication region encodes traits not essential to the bacterial host.

  • Antiobiotic resistance is one of these traits.


History of recombinant dna technology
History of Recombinant DNA technology

In 1971 Cohen, exploited the antibiotic resistance of the plasmids to selectively enrich offspring that contained cell propogating plasmids.

In the late 60s, it was shown that CaCl2 made the cells of E.coli permeable so that they could take up DNA, but could not grow E.coli cells with genetic property changes.

In late 1972, Berg reported on methods for joining fragments of DNA outside of cells using ligases.

Endonucleases, or restrictions enzymes, would however, provide the tool for linking DNA.


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Una cerveza y ...

In Nov. 1972, Berg, Boyer and Cohen met up at a deli bar in Honololu, and discussed the endonuclease that Boyer was working on, and that night they

dreamed of the collaborative project that would be the true start of recombinant DNA technology. In March 1973, the pair produced DNA fragments using Boyer’s technique and joined them to plasmids using Berg’s technique, and then introduced them into bacteria using Cohen’s technique.

The first demonstration of DNA cloning had been achieved.




Recombinant DNA technology

  • The two essential elements of recombinant DNA technology are:

  • 1. Restriction endonucleases

  • 2. Vectors for gene cloning



What 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 the particular sequence recognised by the restriction enzyme


What is a restriction enzyme?

  • Restriction enzymes (or endonucleases) are bacterial enzymes that cut DNA at very specific sequences

  • They generally cut in a ‘staggered’ manner, leaving sticky ends but some enzymes generate blunt ends (i.e. Cut DNA in the middle)

  • Their biological function is to destroy invading foreign DNA


What is a restriction enzyme?

  • Each bacteria has different restriction enzymes

    • Enzymes from E.coli cells cut GAATTC/CTTAAG

    • Enzymes from B. Amyloloquefaciens cut GGATCC/CCTAGG

  • The restriction enzymes are named after the organism from which they were derived

    • EcoRI from E.coli

    • BamHI from B. Amyloloquefaciens


What is a restriction enzyme?

  • Restriction enzymes are used to make recombinant DNA and gene cloning and genetic engineering were made possible by these enzymes

  • Over 200 different restriction enzymes are commercially available (some are VERY expensive)

  • DNA ligase ‘sticks’ the ends back together



What is a restriction enzyme?

  • Recombinant DNA technology can be used to isolate a genomic clone from DNA or for the isolation of human cDNA

  • Isolating a genomic clone provides a piece of DNA identical in base sequence to the corresponding stretch of DNA in the cell and is often designed to contain a specific gene

  • Isolating human cDNA is used for gene expression. Human cDNA (c=complementary) is double stranded DNA copy of mRNA but WITHOUT introns


Vectors for gene cloning


Vector 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


Vector types

  • Plasmids (upto about 20kb insert)

  • Bacteriophage

    •  vectors

  • Can 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


What is a plasmid?

  • Plasmids are small, extrachromosomal pieces of bacterial DNA that are often antibiotic resistant

  • They are ‘shuttle vectors’ to create, produce, and maintain recombinant DNA

  • An example of one of the first plasmids is pBR322

    • Both Amp & Tet resistant, Several unique restriction sites

  • pUC18 now the most commonly used

  • Derivative of pBR322

    • Smaller, Higher copy number per cell, Multiple cloning sites



lacZ gene

  • Gene encoding for enzyme -galactosidase

  • Polylinker resides in the middle

  • Enzyme activity can be measured as marker of gene insertion

    • Disrupted gene – nonfunctional – WHITE

    • Intact gene – functional – BLUE

  • Amp resistance gene still present, Tet resisitance gene omitted



Lambda vector

  • Bacteriophage lambda () infects E.coli

  • Double stranded linear DNA vector, suitable for library construction

  • Can accomodate large segments of foreign DNA, central 1/3 is a ‘stuffer’ fragment

    • Can be substituted with any DNA fragment of similar size

    • Can accomodate  15kbp of foreign DNA



Vectors 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 maintain chromosome in budding

  • - yeast (like , end up as a linear molecules)

  • - a yeast origin of replication, a centromere, and a telomere at each end.

  • - Can clone >2,000 kb (2 Mb).

  • Agrobacterium Tumefaciens

  • Plants

  • Baculovirus

  • Insect cells







Insertion into a Plasmid can be Detected by Disruption of -gal

  • Only bacteria which have taken up plasmid grow on ampicillin.

  • Blue-white selection:

    • white colonies have insert

    • blue colonies have no insert

  • To see blue color, add IPTG (an inducer of -galactosidase expression) and Xgal substrate.


Recombinant DNA technology

  • Class 6 Video 1

  • Class 6 Video 2


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