DAIRY BIOTECHNOLOGY

Module 3. Genetic engineering technology / recombinant DNA technology

Lesson 9
MOLECULAR CLONING - TOOLS

9.1 Introduction

Molecular cloning also referred to as ‘Gene cloning’, ‘Gene Manipulation’, ‘Genetic Engineering’ or ‘Recombinant DNA technology’ involves construction, creation or designing of a recombinant DNA molecule or a new combination of genetic material by constructing in laboratory under controlled conditions of digestion and ligation. DNA from two sources is digested and ligated together to generate a recombinant as shown in Fig. 9.1

Initially, before the invention of PCR, the genomic libraries were prepared to fish out the gene of interest based on functional assays from the resource DNA material (chromosomal or plasmid) using restriction endonucleases either frequent cutters or rare cutters. Currently, the protocol used for gene cloning in vogue is based on amplification of the target gene. This can be brought about by designing suitable primers, if gene sequences are already known and available in the NCBI database. The amplified gene product then can be digested with the appropriate restriction endonucleases and ligated in a vector for propagation in the desired host.

9.2 Basic Steps involved in Molecular Cloning

Basic components involved in molecular cloning are as follows:

1. DNA to be cloned i.e. foreign or insert or passenger or target DNA2. Vector DNA (Vehicle / Carrier for transmission) which includes
Plasmids
Phages like lambda and M-13 viruses
Cosmids
2. Restriction digestion of both target and vector DNA
3. Construction of recombinant DNA (Chimera) i.e. ligation of foreign DNA with vector DNA
4. Introduction of recombinant DNA into host cells of E. coli or yeast or mammalian system by transformation/electroporation
5. Screening and selection of recombinants on the basis of antibiotic resistance or by expression of the target gene
The strategy used to produce recombinant DNA is shown in Fig. 9.2

The following tools are required for carrying out the above steps for construction of a recombinant DNA molecule.

Biological Scissors - Restriction Endonucleases
Vehicle - Plasmid, Phage or cosmid
Glue - DNA Ligase
Sieve - Agarose Gel electrophoresis

9.2.1 DNA to be cloned i.e. foreign or insert or passenger or target DNA

The DNA to be cloned is known as target or foreign or passenger or insert DNA which is obtained from the prokaryotic or eukaryotic source either by digesting with restriction enzymes or by PCR amplification of the target gene using primers designed against the target gene. The PCR amplified product is then digested with the specific restriction enzyme/s to generate compatible ends as that of the vector DNA. The restriction endonucleases will be discussed in the next chapter.

9.2.2 Vector DNA (Vehicle for transmission)

Vector DNA is the vehicle / carrier which is used for carrying the insert / target DNA into the host cell. Vectors will be dealt in detail in a subsequent section.

9.2.3 Construction of recombinant DNA

The recombinant DNA can be constructed using DNA ligase. DNA ligase is an enzyme that joins two DNA fragments by formation of phosphodiester bond between 5’phosphate and 3’ hydroxyl groups in duplex DNA as previously shown in Fig. 9.1. DNA ligase seals the nicks produced in single stranded DNA and can also join cohesive or blunt ends. DNA ligases are of the following two types.

9.2.3.1 E. coli DNA Ligase

E. coli DNA ligase is obtained from an over producing strain of E. coli in which ‘lig’ gene has been cloned. Generally, it is used for joining of cohesive or staggered ends. This enzyme uses NAD as a cofactor.

9.2.3.2 T4 DNA Ligase

T4 DNA ligase was initially purified from phage infected cells of E. coli and is the product of gene 30 of phage T4. Now, it is produced from an over producing strain of E. coli wherein phage T4 gene 30 has been cloned. It uses ATP as a cofactor. T4 ligase can join both cohesive as well as blunt ended DNA fragments.

9.2.4 Introduction of recombinant DNA into host cells of E. coli or yeast or mammalian system by transformation/electroporation

The recombinant DNA is introduced into an appropriate host such as E. coli by calcium chloride induced transformation or electoporation. Both the processes will be discussed in the next sections.

9.2.5 Screening and selection of recombinants on the basis of antibiotic resistance or by expression of foreign DNA

The recombinants / transformants in E. coli can be selected on Luria Bernati (LB) agar supplemented with ampicillin on the basis of ampicillin resistance / inactivation in the vector DNA or any other antibiotic resistance or phenotypic marker. Another way of selecting recombinants is on the basis of detection of the recombinant protein expressed in the transformants or the recombinant clones.

The presence of the recombinant vector in the transformants can be monitored on agarose gel after its isolation from the recombinant clones by electrophoresing them on running agarose gel.

9.3 Agarose Gel Electrophoresis

Agarose gel electrophoresis is a widely used method that separates molecules based upon charge, size and shape and is used for separating charged biomolecules such as DNA, and RNA. Agarose is a polysaccharide extracted from a seaweed (Gelidium genera of seaweed). It is a linear polymer made up of repeated subunits of D-galactose and 3,6, anhydro-L-galactose. Most agarose gels are made using 0.7% (good for separation of large size DNA fragments of 5–10 kb) and 1- 2% (for small 0.1–1.0 kb fragments). The commonly used buffers used in the agarose gel electrophoresis for separation of the recombinant DNA molecules include TAE (Tris Acetate EDTA), TBE (Tris Borate EDTA) and TPE (Tris Phosphoric acid EDTA). However, the most extensively used buffer in majority of laboratories is TAE. Prior to gel electrophoresis, the DNA samples are mixed with xylene cyanol and bromophenol blue which are the most commonly used DNA loading buffers / dyes in order to monitor the movement of DNA samples.

9.3.1 Procedure

Generally, agarose of 0.7 – 2.0 % is prepared depending on size of DNA by dissolving the appropriate quantities of agarose in 1 X TAE buffer. Ethidium bromide stock solution (10 mg/ml) is added directly to molten agarose solution at the rate of 0.5 µg/ml before casting the gel. Molten agarose is then cooled to 50oC and poured on to respective moulds (mini or maxi) with appropriate combs. The series of steps are illustrated in Fig. 9.3. However, before pouring the gel, the surface of the mould should be leveled properly. After complete setting of the gel, the comb is removed carefully and gel plate is mounted on to the respective electrophoresis apparatus filled with 1 X TAE buffer. The DNA samples are mixed with tracking dye and loaded slowly into the wells using a micropipette. Electrophoresis is carried out at 80 V till the tracking dye reaches one third of the length of the gel. After completion of the electrophoresis, the gel is examined under UV trans-illuminator and photographed using Gel documentation systems (Bio-Rad, Minibis, G-box etc.).

Books

From Genes to Genomes: Concepts and Applications of DNA Technology, 3^rd Edition, Jeremy W. Dale, Malcolm von Schantz, Nicholas Plant (Eds), Wiley-Blackwell, 2011, ISBN: 978-0-470-68386-6

Microbial Genetics, 2^nd Edition, Stanly R Maloy, John Cronan, David Freifelder, Narosa, ISBN: 8173196974

Molecular Biology of the Gene, Sixth Edition, James D. Watson (Editor) Cold Spring Harbour Press and Benjamin Cummings, ISBN 978-080539592-1

Molecular Biotechnology - Second Edition, S. B. Primrose, Blackwell Science Inc., ASIN: 0632030534

Introduction to Biotechnology, Brown, C.M., Campbell, I and Priest, F.G. Panima Publishing Corporation, 2005. ISBN : 81-86535-42-X

DNA and Biotechnology, Fitzgerald-Hayes, M. And Reichsman, F. 2^nd Amsterdam : Elsevier, 2010. ISBN : 0-12-048930-5

Molecular Biotechnology : Principles and Applications of Recombinant DNA, Glick, B.R., Pasternack, Jack, J and Patten, Cheryl, L (Eds)., 4th Washington., ASM Press, 2010. ISBN : 1-55581-498-4

Molecular Biology and Biotechnology : a guide for students, Krauzer, H. And Massey, A.(Eds) 3^rd Washington DC : ASM Press, 2008, ISBN : 978-155581-4724

Recombinant DNA and Biotechnology : a guide for teachers, Kreuzer, H and Massey, A. 2^nd Washington : ASM Press, 2001, ISBN : 155581-175-2

Molecular Biotechnology, Primrose, S.B. 2nd New Delhi : Panima, 2001. ISBN : 81-86535-21-7

Molecular Biology and Biotechnology, Smith, C.A. and Wood, E.J. London : Chapman and Hall, 1991.ISBN : 0-412-40750-7

Introduction to Biotechnology, Thieman, W.J and Pallidino, M.A. 2^nd New York : Pearson, 2009, ISBN : 978-0-321-58903-3

Molecular Biology and Biotechnology, Walker, J.M and Rapley, R. 4^th – New Delhi : Panima Publishing Corporation, 2003, ISBN : 81-86535-40-3

Gene Biotechnology, Wu William, Welsh, M.J., Kaufman, P.B and Zhang, H.H. 2^nd Boca Raton : CRC press, 2004. ISBN : 0-8493-1288-4

Internet Resources

http://en.wikipedia.org/wiki/Molecular_cloning

http://www.blackwellpublishing.com/allison/docs/sample_ch8.pdf