Tools of Genetic Engineering 2

1. Tools of Genetic Engineering 2 By: Russel Jean Gallo

2. EnzymesA detailed account of enzyme technology is given in (Enzyme Technology). Apart from this, there are many enzymes which are used in genetic engineering as an important biological tool, some of them are described below: • ExonucleasesThese enzymes act upon genome and digest the base pairs on 5' or 3' ends of a single stranded DNA or at single strand nicks or gaps in double stranded DNA (Fig. 3.1A). • EndonucleasesThey act upon genetic material and cleave the double stranded DNA at any point except the ends, but their action involves only one strand of the duplex (Fig. 3.1B). • Restriction Endonucleases • These enzymes occur naturally in bacteria as a chemical weapon against the invading viruses and cut both strands of DNA when certain foreign nucleotides are introduced in the cell. These enzymes cleave a DNA to generate a nick with a 5' phosphoryl and 3' hydroxyl termini. There are two major restriction enzymes: Type I and Type II. Type f enzymes recognize a specific sequence of DNA molecule but cut elsewhere, and Type II make cuts only within the restric­tion sites and produce two single strand breaks, one break in each strand. Type II enzymes are the most impor­tant ones.

3. As a result of their action the broken nucleotides form a DNA duplex which exhibit two fold symmetry around a given point. In some cases, cleavage in two strands are staggered to produce sin­gle stranded short projections opposite to each other with blunt or mutually cohesive sticky ends which are identical and complementary to each other (Fig. 3.1C). • These complementary sequences are also known as palindrome sequences or palindromes. Therefore, when read from 5' 3' both strands have the same sequence (Glover, 1984; Boffey, 1987). Nowadays a large number of restriction enzymes are available commercially (Table 3.1). Some of the commonly used restriction endonucleases are given in Table 3.2.Roberts (1983) has given an extensive list of restriction enzymes and the sequences recognized by them. Most of the enzymes share a common central nucleotide in their recognition sequence for example, BamHI, BglII, Sau3A. But these enzymes recognize different sites in DNA and produce identical single stranded 5' tails which allow the joining of fragments generated by different enzymes within this set. The identical nature of the termini of cleaved DNA fragments from any organism is the very property which permits the annealing and subsequent ligation of the DNA from diverse sources (Glover, 1984).

4. Foreign DNA / Passenger DNAForeign/passenger DNA is a fragment of DNA molecule which is enzymatically isolated and cloned. The gene is identified on a genome and pulled out from it either before or after cloning. The cloned foreign DNA fragment expresses normally as in parental cell. Thus, the foreign DNA fragments can be procured from a variety of sources depending on the aims and scope of cloning experiments. • Identification and characterization of DNA sequences are rather more difficult on its genome than using mRNA, if it is in pure form. If the gene product translated by mRNA is not well characterized it can be most difficult procedure for cloning. In an average cell or tissue, 1-2% of total cytoplasmic RNA population is mRNA which carry transcripts for coding various proteins. When mRNA is present in low amount it is rather difficult to isolate cDNA clones. The procedures of isolation and purification of mRNA and getting pure cDNA are given in Genes : Nature, Concept and Synthesis. • Besides cDNA, clonable DNA fragments are also isolated from the donor organisms by using restriction endonucleaees or can be procured from a gene bank (see Figure below) constructed for the same purpose.

5. Cloning VectorsVectors are those DNA molecules that can carry a foreign DNA fragment when inserted into it. Vectors are also known as vehicle DNAs. Based on the nature and sources, the vectors are grouped into bacterial plasmids, bacteriophages, and cosmids and phasmid. • PlasmidsPlasmids are the extrachromosomal, self-replicating, and double stranded closed and circular DNA molecules present in the bacterial cell. Plasmids contain sufficient genetic informations for their own replication. A number of host properties are specified by plasmids, such as antibiotic and heavy metal resistance, nitrogen fixation, pollutant degradation, bacteriocin and toxin piroductioivcolicin factors and phages (Dahl et al, 1981). Naturally occurring plasmids can be modified by in vitro techniques. Cohen et al. (1973) for the first time reported the cloning DNA by using plasmid as vector.

6. cDNA Clone Bank or cDNA Library • True copy of an mRNA molecule is known as copy DNA or cDNA. The well characterized cDNA molecule is allowed to bind with a suitable vector which then transforms a bacterial cell in such a way that (Joes not disrupt its normal func­tion. The transformed bacterial cell con­taining plasmid with DNA copy of an mRNA molecule is known as cDNA clone. lt is difficult to have cDNA from double stranded DNA molecules. There­fore, most of the cDNA clones have been prepared from mRNA sequences of eukaryotic cells. The procedure for obtain­ing cDNA to built a library (Fig. 4.1) is given under cDNA to be cloned (seeIsolation of DNA to be cloned). • A typical eukaryotic cell contains between 10,000 to 30,000 different mRNA sequences. Williams (1981) has defined the cDNA clone bank as “a population of bacterial transformants, each containing a plasmid with a single cDNA insert, and with a sufficiently large number of individual transformants such that every mRNA molecule is represented at least once in the bacterial population”. A complete bank is that which contains in the order of 5,000 to 10,000 clones of the sequences amenable to detection.

7. Gene Bank or Genomic Library • Gene bank or genomic library is a complete collection of cloned DNA fragments which comprises the entire genome of an organism (Dahl et al., 1981). Gene bank is constructed by a shotgun experiment where whole genome of a cell is cloned in the form of random and unidentified clones. The clones of DNAs are produced by (i) isolation of DNA fragments to be cloned, (ii) joining the fragments to a suitable vector (usually phage 1), (iii) introduction of recombinant DNA into host cells at high efficiency to get a large number of independent clones, (iv) selection of the desired clones, and (v) use of clones for the construction of gene bank (Fig. 3.9).Size fractionation of DNA (into about 10 Kb or more in size) to be cloned is important in approaching the maximum cloning capacity of the vector used. However, DNA fragments of a size not suitable for cloning will be ligated to vector and will lower the efficiency of introduction of the recombinant DNA into cells. DNA fragments of the suitable sizes can be obtained through particle digestion with several restriction enzymes and by agarose gel electrophoresis gradient centrifugation (Dahl et al, 1981). These fragments are cloned without any attempt to select for particular sequences. Bacteria containing recombinant DNA are plated to give rise individual colonies. Each colony contains a fragment of genome. If the size of the fragments is small, more colonies are needed to be grown just to be sure for the presence of any particular gene. It has been calculated that for 10 Kb fragment, about 1.5 x 103 colonies are required for the E.coligenome and about 2 x 106 colonies forHomo sapiens (Boffey, 1987).

8. Electrophoresis-electrophoresis refers to carrying something by applying electricity. It is an analytical device commonly used for separation and purification of DNA fragments. A gel is used in electrophore­sis which is either polyacrylamide or agarose.The former is preferred for smaller DNA fragments and the latter for larger ones. Agarose is a purified powder isolated from agar, a gelatinous material of sea weeds. Agarose powder when dissolved in water and boiled results into gel form. The gel prepared in a mixture of salt and water becomes a goods conductor of electricity. The gel forms small pores the size of which varies depending on its amount in a given water. These pores act as molecular sieve. These allow the larger molecules to move more slowly than the smaller molecules.

9. the electrophoresis box consists of a positive and a negative electrode, a shelf designed to held the gel, a comb used to form the wells within the gel, and a power supply (Fig. 3.10A). The DNA to be electrophoresed is digested with restriction enzymes which yields DNA fragments of unequal length. The fragments are mixed with sucrose and a dye (ethidium bromide or methylene blue) which altogether is known as loading dye. Sucrose increases the density of DNA prepara­tion and dye increases the visibility of the preparation.

10. The preparation is loaded into wells at one end of the gel. At least one well is filled with reference DNA (i.e. DNA fragments of known length) for comparison with those of unknown length. Electric current is applied at opposite ends of electrophoresis chamber. A current is generated between a negative electrode at the top of loading end of the gel and a positive electrode at the bottom of the end of gel resulting in movement of fragments through pores of the gel. DNA molecules have a negative electric charges due to PO4-4 which alternate with sugar molecules. Opposite electric charges tend to attract one another. The small DNA molecules move at faster speed as compared to larger ones. All DNA molecules of a given length migrate nearly the same distance into the gel and form bands. Each band represents many copies of DNA fragments having about the same length. After completion of electrophoresis gel is removed from the chamber and stained to make bands easily seen either with ethidium bromide (EB) or methylene blue. When gel is illuminated with UV light, fluorescent orange, bands appear due to EB; methylene blue results in blue bands under normal room temperature (Fig. 3.10B).