Bio 423 Lecture 4

BB211: Cell and Molecular Biology Dr Eve Lutz Department of Bioscience

Recombinant DNA technology: Lecture 4 Making & Screening Libraries: Selection of a specific clone from a pool of recombinants References for lectures on recombinant DNA technology please read the following: Chapter 16 Klug, WS & Cummings, MR Essentials of Genetics, 4th ed.

What is a DNA library and why construct one?

A DNA library consists of cloned DNA fragments representing the entire genome of an organism (genomic DNA library) or of the protein-encoding genes only (cDNA library). Genomic library: sequence cDNA library:

cDNA

Contains entire DNA content of an organism Suitable for determining genomic DNA Requires chromosomal DNA isolation Contains entire protein-encoding DNA content Messenger RNA used as a starting material Messenger RNA reverse-transcribed into Requires mRNA isolation

Isolating and replicating sections of the chromosomal DNA of an organism in order to examine its properties (e.g. DNA sequence,

sequence of protein encoded). DNA is replicated after cloning into vectors - usually plasmids or viruses that can exist in multiple copies in cells.

Choosing the appropriate library 1) cDNA or genomic? Do we want to obtain fragments of the complete genome e.g. to get DNA sequence, look at promoter regions, see what introns are in DNA (genomic library). Or do we want to clone protein-encoding genes and use them for protein expression (cDNA library). 2) What vector system? What sizes of inserts do we want to clone? Do we want to be able to generate proteins directly? Do we want to be able to produce single-stranded DNA and sequence it directly? 3) Which host system to use? Usually either Escherichia coli or Saccharomyces cerevisiae.

Having created a gene library; how do we identify a desired recombinant? If we wish to identify a specific recombinant from a gene library, and we know some of the sequence of that gene, then a good method to use is DNA hybridisation. However, in many cases we may not have this information. Another way of identifying recombinants is to use expression screening to look for the production of protein.

Selecting the probe If the gene or similar sequence has been cloned from another cell type or species then it is possible to use that cDNA or genomic DNA fragment or an oligonucleotide derived from the gene sequence. If the protein of interest has been isolated, then it

may be possible to raise antibodies against it, or to sequence it in order to derive the gene sequence from the amino acid sequence see how to design degenerate probes. Otherwise some sort of functional assay may have to be used to detect expression of the desired gene.

Plate out library Gene libraries can contain millions of different clones and it is necessary to have a means of isolating positive clones from the rest of the collection. Plasmid libraries (in E. coli transformants) are often plated on to agar plates containing the selection antibiotic to allow colonies to grow. Phage libraries are incubated with E. coli, then plated into soft agar to allow plaques to form. Once a library is plated onto agar, nitrocellulose filters or nylon membranes are laid on the surface of the plates. Plaques or colonies are 'lifted'when the filter/membrane is carefully peeled off the plate. The filter is a replica of the original plate. Bacterial cells within colonies are 'broken' to release their contents by soaking membrane in detergent (SDS) and protease. For phage, the cells have already been lysed and DNA is exposed. In both cases, DNA is denatured with alkali and bonded firmly to the membrane by baking at high temperature or using UV light to cross-link it to the filter. Several rounds of isolation may be required to purify individual clones Large libraries are usually plated at high density, so it is difficult if not impossible to pick individual positive spots or clones. Mixture of clones from first pick can be replated at lower density and reprobed to pick out individual positives

DNA hybridisation This is achieved by labelling a small probe - often by incorporating radioactivity into it (see also Southern Blotting). For instance, if we chemically synthesise an oligonucleotide, of the same sequence

as the gene of interest, this can be labelled with a radioactive phosphate (32P) from radiolabeled ATP. This is done by removing the 5' phosphate of the oligonucleotide using alkaline phophatase, and replacing it with a radiolabelled phosphate using polynucleotide kinase.

Fig. 7-20, Lodish et al. (4th ed.) An alternative is to incorporate one or more radiolabeled nucleotides to the 3' end of the probe using terminal transferase. Oligonucleotide probes can be as small as 20 nucleotides. It is important to remember that the DNA probe must be specific for the gene of interest and should not cross-react with other recombinants - that is anneal to sequences present in other clones. The probability of a specific 20 nucleotide sequence occurring is very low, i.e. 1 in 420 (1 in ~1012) nucleotides. The membrane is soaked with a solution of the radiolabeled probe, to allow it to hybridise to the correct gene. The membrane is washed extensively to remove non-specifically bound probe, and the colonies/plaques to which the probe remains bound are visualised by autoradiography.

Plaque hybridisation

Fig. 7-18, Lodish et al. (4th ed.)

Expression screening This is frequently done using libraries in phage λ expression vectors such as λgt11. Target proteins are produced as fusion proteins wit beta-galactosidase where the insert DNA is cloned into the lacZ complex which is present in the lambda gt11 vector DNA. Expression can then be switched on using IPTG. In a similar method to the DNA hybridisation method, proteins expressed from the library are bound to membranes or filters. The filters/membranes are then incubated with a specific antibody to detect the desired protein.

Fig. 7-21, Lodish et al. (4th ed.) There are many different expression screening methods that can be used to isolate a particular gene, and are designed according to what is known about the protein function. Sometimes it is necessary to be able to clone adjacent pieces of DNA. A technique known as chromosome walking is used to move systematically along a chromosome from a known location and to

clone overlapping genomic clones that represent progressively longer parts of a particular chromosome. Chromosome walking is used as a means of finding adjacent genes (positional cloning), or parts of a gene which are missing in the original clone as well as to analyse long stretchs of eukaryotic DNA webpage last updated 20/2/03 Back to Recombinant DNA Technology Lecture List