Biotechnology - Recombinant DNA Technology PDF

11.2 TooLS OF RECOMBINANT DNA TECHNOLoGY Now we know from the foregoing discussion that genetic engineering or recombinant DNA technology can be accomplished only if we have the key tools, i.e., restriction enzymes, polymerase enzymes, ligases, vectors and the host organism. Let us try to understand...

11.2 TooLS OF RECOMBINANT DNA TECHNOLoGY Now we know from the foregoing discussion that genetic engineering or recombinant DNA technology can be accomplished only if we have the key tools, i.e., restriction enzymes, polymerase enzymes, ligases, vectors and the host organism. Let us try to understand some of these in detail. 11.2.1 Restriction Enzymes In the year 1963, the two enzymes responsible for restricting the growth of bacteriophage in Escherichia coli were isolated. One of these added methyl groups to DNA, while the other cut DNA. The later was called restriction endonuclease. The first restriction endonuclease-Hind I, whose functioning depended on a specific DNA nucleotide sequence was isolated and characterised five years later. It was found that Hind II always cut DNA molecules at a particular point by recognising a specific sequence of six base pairs. This specific base sequence is knoWn as the recognition sequence for Hind I. Besides Hind II, today we know more than 900 restriction enzymes that have been isolated from over 230 strains of bacteria each of which recognise different recognition sequences. The convention for naming these enzymes is the first letter of the name comes from the genus and the second two letters come from the species of the prokaryotic cell from which they were isolated, e.g., EcoRI comes from Escherichia coli RY 13.In EcoRI, the letter 'R is derived from the name of  strain. Roman numbers following the names indicate the order in which the enzymes were isolated from that strain of bacteria. Restriction enzymes belong to a larger class of enzymes called nucleases. These are of two kinds; exonucleases and endonucleases. Exonucleases remove nucleotides from the ends of the DNA whereas, endonucleases make cuts at specific positions within the DNA. Each restriction endonuclease functions by 'inspecting the length of a DNA sequence. Once it finds its specific recognition sequence, it willbind to the DNA and cut each of the two strands of the double helix at specific points in their sugar-phosphate backbones (Figure 11.1). Each restriction endonuclease recognises a specific palindromic nucleotide sequences in the DNA. Action of Restriction enzyme The enzyme cuts both DNA EcoRI cuts the DNA between bases6/ 16 strands at the same site G and A only when the sequence GAATTC is present in the DNA Vector DNA Foreign DNA EcoR1 Sticky end Sticky end DNA fragments join at sticky ends Recombinant DNA 11.1 Steps in formation of recombinant DNA by action of restriction endonuclease enzyme - EcoRI Do you know what palindromes are? These are groups of letters that form the same words when read both forward and backward, e.g., "MALAYALAM". As against a word-palindrome where the same word is read in both directions, the palindrome in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept the same. For example, the following sequences reads the same on the two strands in 5> 3 direction. This is also true if read in the3 5direction. 5 GAATTC 3 3 CTTAAG 5' Restriction enzymes cut the strand of DNAalittle away from the centre of the palindrome sites, but between the same two bases on the opposite strands. This leaves single stranded portions at the ends. There are overhanging stretches called sticky ends on each strand (Figure 11.1). These are named so because they form hydrogen bonds with their complementary cut counterparts. This stickiness of the ends facilitates the action of the enzyme DNA ligase. Restriction endonucleases are used in genetic engineering to form 'recombinant' molecules of DNA, which are composed of DNA from different sources/genomnes. When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of 'sticky-ends' and, these can be joined together (end-to-end) using DNA ligases (Figure 11.2). Vector Foreign DNA DNA (plasmid) Same restriction enzyme cutting both foreignb DNA and vector DNA at specific point Ligases join foreign DNA to plasmid Transformation E.coli Cells divide Figure 11.2 Diagrammatic representation of recombinant DNA technology  You may have realised that normally, unless one cuts the vector and the source DNA with the same restriction enzyme, the recombinant vector molecule cannot be created. Separation and isolation of DNA fragments : The cutting of DNA by restriction endonucleases results in the fragments of DNA. These fragments can be separated by a technique known as gel electrophoresis. Since DNA fragments are negatively charged molecules they can be separated by forcing them to move towards the anode under an electric field through a medium/matrix. Nowadays the most commonly used matrix is agarose which is a natural polymer extracted from sea weeds. The DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel. Hence, the smaller the fragment size, the farther it moves. Look at the Figure l1.3 and guess at which end of the gel the sample was loaded. The separated DNA fragments can be visualised only after staining the DNA Wells with a compound known as ethidium DNA bands bromide followed by exposure to UV Largest Smallest radiation (you cannot see pure DNA fragments in the visible light and without staining). You can see bright orange coloured bands of DNA in a ethidium bromide stained gel exposed to UV light (Figure 11.3). The separated bands of DNA are cut out Figure 11.3 A typical agarose gel from the agarose gel and extracted electrophoresis showing from the gel piece. This step is known migration of undigested as elution. The DNA fragments (lane l) and digested set of purified in this way are used in DNA fragments (lane 2 to 4) constructing recombinant DNA by joining them with cloning vectors.