topics in (nano) biotechnology lecture iii 10th april phd course

TOPICS IN (NANO) BIOTECHNOLOGY

Lecture III

10th April

PhD Course

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

• This week we will look at Recombinant DNA technology

• We will also look at the amplification of DNA and finally at its sequencing

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

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

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

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

Una cerveza y ...In Nov. 1972, Herb 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 and joined them to plasmids using Boyer’s technique, and then introduced them into bacteria using Cohen’s technique.

The first demonstration of DNA cloning had been achieved.

But let’s look at it in more detail....

Recombinant DNA technology

Recombinant DNA technology

• The two essential elements of recombinant DNA technology are:

1. Restriction endonucleases2. Vectors for gene cloning

Restriction endonucleases

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?

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• Antibiotic resistance genes and/or other

selectable markers• Examples of cloning vectors are

• plasmid• bacteriophages • yeast artificial chromosomes (YAC)• bacterial artificial chromosomes (BAC)• retrovirus

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 – WHITEWHITE• Intact gene – functional – BLUE

• Amp resistance gene still present, Tet resisitance gene omitted

What is a bacteriophage?

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

Recombinant DNA technology

Recombinant DNA technology

Recombinant DNA technology

Video 3a: Plasmid Cloning

• Genes can be cloned in recombinant DNA vectors

• Cloning vector

• Procedure for cloning a eukaryotic gene in a bacterial plasmid

1. Isolation of vector and gene-source DNA2. Insertion of DNA into the vector3. Introduction of cloning vector into bacterial cells4. Cloning of cells (and foreign gene)5. Identification of cell clones carrying the gene of interest

• Nucleic acid hybridization• Nucleic acid probe

Genomic Clones

Genomic Clones

Genomic Clones

cDNA Clones • Genes can be cloned in recombinant DNA

vectors• Cloning vector• Procedure for cloning a eukaryotic gene in a

bacterial plasmid• Cloning and expression eukaryotic genes:

problems and solutions

1. Difference in promoters

Expression vector

2. Introns

Complementary DNA (cDNA)

cDNA Clones

• Cloned genes are stored in DNA libraries1. genomic library – cloned set of rDNA fragments

representing the entire genome of an organism

2. cDNA library - cloned set of rDNA fragments representing genes transcribed in a particular eukaryotic cell type (no introns, extrons etc)

• rDNA fragments generated, ligated & cloned

• The larger the fragments that are cloned, the smaller the size of the library

Genomic and cDNA Libraries

• Contains at least 1 copy of each fragment

• Screened using nucleic acid probes to identify specific genes

• Subcloning usually necessary for detailed analysis of genes

• N = ln (1-P)/ln (1-f)e.g. Human genome = 3.2 x 109bp

Lambda vector can accommodate 17kbp inserts

N = ln(1-0.99)/ln(1-(1.7x104bp insert/

3.2 x 109bp genome))

N = 8.22 x 105 plaques required in library

Genomic Libraries

• mRNA represents genes that are actively transcribed (or expressed)

• Eukaryotic mRNA – introns have been removed

• mRNA – converted into a DNA copy (cDNA)

• Size of library depends on number of ‘messages’

• More complex than genomic library

cDNA Libraries

Genomic Libraries

• Libraries searched using specific probe• Specificity extremely important• Single-stranded nucleic acid fragments

• Radioactive vs non-radioactive• Radioisotopes serve as tag - autoradiography• Chemiluminescence, colorimetric, fluorescence

• Sources of probes• Heterologous (other species)• cDNA (genomic sequences with introns/promoter

elements)• Probe based on protein sequence

• 18-21 bases sufficient (ssDNA, RNA, antibodies)

ID of specific DNA sequences

• Expression Library• Detect protein product of clone using antibodies• Microarray technology

ID of specific DNA sequences

• Chromosome walking

•If nearby sequences have been cloned, this can be used as starting point for isolation of adjacent genes

• The PCR clones DNA entirely in vitro

• Polymerase chain reaction1. Denaturation (heat to ~94oC)

2. Annealing (37-72oC)

3. Extension (72oC)

Polymerase Chain Reaction

Polymerase Chain Reaction

Video

• Separation of DNA fragments based on size, charge and shape differences

• Standardised MW markers run on the same gel for size comparison

Agarose gel electrophoresis

Gel electrophoresis

Video

• DNA digested with restriction enzymes and separated by gel electrophoresis

• Gel treated with NaOH to denature DNA to ssDNA

• DNA transferred from gel to DNA binding filter

• DNA ‘fixed’by baking membranes/UV

• Incubate with ssDNA probe

• Autoradiography/chemiluminescence

Southern blotting

Southern Blotting

DNA sequencing

Isolation, amplification & sequencing

3 videos

Exercises • Describe a plasmid prep.• What is meant by the term ‘sticky ends’?• What is a genomic clone?• What is a cDNA clone?• What are the steps involved in making a genomic library?• Explain in a few sentences the importance & principle of PCR.• Explain in a few sentences the importance & principle of gel electrophoresis.• Explain in a few sentences the importance & principle of southern blotting.• Explain in a few sentences the importance & principle of DNA sequencing.