TOPICS IN (NANO) BIOTECHNOLOGY

Lecture 7

5th May, 2006

PhD Course

• 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

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

• Class 7_ Video 1

Polymerase Chain Reaction

• Class 7_ Video 1a

• 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

DNA sequencing

Sequencing_movie_1 Sequencing_movie_2

DNA sequencing http://www.dnalc.org/shockwave/cycseq.html

Isolation, amplification & sequencing

• Class 7_Isolation

• Class 7_Amplification

• Class 7_Sequencing