Bio 423 Lecture 3

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

Recombinant DNA technology: Lecture 3 Recombinant DNA: Plasmids, cloning Background reading: Reference for this lecture please read the following: Chapter 16 Klug, WS & Cummings, MR Essentials of Genetics, 4th ed.

What is DNA cloning? DNA cloning is the isolation of a fragment or fragments of DNA from an organism and placing in a VECTOR that replicates independently of chromosomal DNA. The RECOMBINANT DNA is propagated in a host organism; the resulting CLONES are a set of genetically identical organisms which contain the recombinant DNA.

Why is DNA cloning important? DNA cloning is involved in a number of applications (GENETIC ENGINEERING). Many techniques for DNA isolation and manipulation have been worked out and are now routine in scientific laboratories. These techniques are now important tools that every scientist must know about.

Three main purposes for cloning DNA 1) DNA sequencing Determining the sequence of the bases in the DNA can tell us

about which proteins or RNAs are encoded and their sequences, which sequences control their expression (GENE PROMOTERS and other control sequences), as well as any possible mutations which might alter their function. Having access to the complete DNA sequence of an organism can help us decipher the biology of that organism. 2) Protein production Isolating the gene which encodes a desired protein (haemoglobin, interferon) may allow that gene to be over-expressed so that the protein can be produced in bulk for study or use 3) Engineering animals/plants/proteins The ability to alter the properties of proteins as well as create genetically modified animals and plants (TRANSGENICS) has lead to their use for research and for therapeutic and commercial purposes. The technology may lead to the development of new therapies for the treatment of disease (GENE THERAPY).

Cloning and Expression Vectors Isolated DNA is cloned into VECTORS for long term storage, propagation of the DNA and for production of protein from gene(s) encoded in the DNA

What are cloning vectors? Cloning vectors are extra-chromosomal 'replicons' of DNA which can be isolated and can replicate independently of the chromosome. Vectors usually contain a selectable marker - a gene that allows selection of cells carrying the vector e.g. by conferring resistance to a toxin. DNA of interest can be inserted into the vector and replicated in host cells, usually one which has been well characterized. Commonly used vector systems • •

Bacterial plasmids Bacteriophages

• • • •

Cosmids Yeast artificial chromosomes (YACs) Ti plasmid (plants) Eukaryotic viruses such as baculovirus (insect cells), SV40 virus and retroviruses.

Plasmids are the most commonly used vector system. Several types are available for cloning of foreign DNA in the host organism Escherichia coli. Many E. coli plasmids allow the expression of proteins encoded by the cloned DNA

Bacteriophage are another common vector system used for

cloning DNA. These are viruses which 'infect' E. coli. The M13 bacteriophage is a single-stranded DNA virus which replicates in E. coli in a double-stranded form that can be manipulated like a

plasmid. It can be used to produce single-stranded DNA copies which are useful for DNA sequencing. Bacteriophage λ is another bacteriophage which is commonly used to make DNA libraries. It allows the cloning of larger fragments of DNA than can be incorporated into plasmids.

Strategy for cloning DNA into a plasmid (or other cloning) vector

SUBCLONING • • • •

cut DNA of interest with the appropriate restriction endonuclease(s) separate fragments by gel electrophoresis purify target fragment from gel ligate fragment with a plasmid cut with the same restriction endonuclease(s) - ligation is performed using the enzyme T4 DNA ligase, ATP and Mg2+ ions

Subcloning an EcoRI fragment into a plasmid cloning vector

Good efficiency of ligation of foreign DNA into a vector can be achieved if both the vector and the insert DNA are cut with 2 different restriction enzymes which leave single stranded ends (cohesive ends). The DNA is ligated in only one direction, and there is only a low background of non-recombinant plasmids. If only one restriction enzyme is used to cut the vector and insert, then efficiency of ligation is lower, DNA can be inserted in two directions and tandem copies of inserts may be found. To avoid high background of non-recombinants, alkaline phosphatase is used to remove 5' phosphate groups from the cut vector to prevent self-ligation.

If vector and insert DNA are cut with an enzyme which leaves blunt DNA ends, the background of non-recombinant plasmids can be high and the best way around the problem is to use high concentrations of both DNAs and of the DNA ligase enzyme. Transformation is the process by which plasmids (or other DNA) can be introduced into a cell. For E. coli transformation with plasmids is quite straightforward, plasmids can be introduced by electroporation or by incubation in the presence of divalent cations (usually Ca2+) and a brief heat shock (42°C) which induces the E. coli cells to take up the foreign DNA There are different methods to select for transformed cells. For instance, transformants can be selected as antibiotic-resistant colonies on agar plates containing antibiotic. For E. coli transformed with plasmids, colonies grown on antibiotic-containing plates should all carry plasmids. However, this does not guarantee that the plasmid contains an insert. It is possible that the vector has re-ligated and not incorporated an insert.

A means to determine which clones contain plasmids with inserts is to use a positive control method such as insertional inactivation. This provides a clear way of recognising recombinants from those carrying re-ligated vector. 1. two antibiotic selection and replica plating

2. colour selection: blue/white selection using the lacz gene

Analysis of clones One of the first steps is to identify clones carrying the recombinant plasmid, with the desired DNA insert. This can be done by 'picking' clones - choosing individual bacterial colonies in order to isolate the plasmid DNA from each of them. Single bacterial colonies are grown in culture broth containing the selection antibiotic in order to maintain the plasmid. The plasmid DNA is extracted by the standard minipreparation technique and then analysed by restriction digest. After digesting the DNA, different sized fragments are separated by agarose gel electrophoresis and the sizes determined by comparison with known DNA molecular weight markers.

Restriction enzymes are a useful tool for analysing Recombinant DNA • • •

Checking the size of the insert Checking the orientation of the insert Determining pattern of restriction sites within insert DNA

Sometimes it is important to determine the orientation of the DNA insert in relation to the vector sequence. This can be done simply by restriction digest using enzyme(s) which cut the vector sequence near to the insert and cut within the insert sequence (asymmetrically).

Figure of agarose gel stained with ethidium bromide and visualised with uv light webpage last updated 20/2/03 Back to Recombinant DNA Technology Lecture List