Lecture 3: January Recombinant DNA Technology and Enzymes

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1 CS Computational Biology for Computer Scientists Spring 2003 Lecturer: Gene Myers Lecture 3: January 28 Scribe: Manikandan Narayanan Disclaimer: These notes have not been subjected to the usual scrutiny reserved for formal publications. They may be distributed outside this class only with the permission of the Instructor. 3.1 Plan DY 1: Introduction to Computational Biology. DY 2: DN, RN, Proteins, Central Dogma (Gene Structure and Expression). DY 3: Recombinant DN Technology and Enzymes, Labelling, Separation, Cloning. DY 4: PCR, CODIS Fingerprinting, DN Sequencing, Sequence Comparison. Today in DY 3, we focus on the techniques/operators that molecular biologists use for manipulating DN in their experiments. There is a need for computational biologists to understand the nature of data and sources of errors in these experiments, and interaction with molecular biologists becomes easy if we are aware of these techniques. book on genomes ([B99]) was used for some of the definitions given in these notes. 3.2 Recombinant DN Technology and Enzymes Recombinant DN technology is about using in vivo (inside a living organism) enzymes in vitro (inside an aqueous watery environment). Following is a list of some important enzymes and the things they do. These enzymes form a library of basic operators that can be used in designing molecular biology experiments. (In the text that follows, stands for single-stranded and d.s for double-stranded.) 1. Polymerase: Polymerase enzymes synthesize new polynucleotides complementary to a given DN or RN template. When the given template is a chain, a d.s DN called the primer is required to start the synthesis. In addition dntp 1 (deoxynucleotide tri-phosphate) molecules, which are energetic molecules trying to get rid of two of their three phosphate groups, are needed as raw materials for the synthesis. Figure 3.1 shows an example synthesis reaction. If needed, the synthesized DN can be disassociated into its two strands by heating it up or by chemical treatment. Depending on the start and end products of the synthesis, there are two types of polymerases. One does (a) Transcription - Given a DN template, synthesizes a new RN, and the other does (b) Replication - Given a DN template, synthesizes a new DN. There is also another type called the reverse transcriptate, which as the name suggests, synthesizes a DN given a RN template. Viruses do reverse transcription to store their RN in DN form before entering dormancy. (Viruses are active when their RN is around.) 1 N is a wild-card that can represent any of the four nucleotides, G, C or T 3-1

2 3-2 Lecture 3: January 28 Polymerase dntp d.s PRIMER 18 bp C G T T G G C C T T DN Figure 3.1: Sythesis of a complementary DN chain by polymerase Ligase C G T Ligase C G T G C G C Figure 3.2: Ligase joining blunt-ends (top) and sticky-ends (bottom) 2. Ligase: Ligases are enzymes that join DN chains. They synthesize the linking bonds using phosphate molecules at the end of DN chains. There are two types of ligases based on the ends of the chains they join. (a) Blunt-end: These type of ligases join DN chains with blunt ends i.e., ends which are doublestranded as shown in Figure 3.2 (top). (b) Sticky-end: These ligases join DN chains that have complementary single-stranded ends called sticky-ends (Figure 3.2 bottom). These are thermodynamically favourable reactions since there is a natural tendency for the complementary ends to come together and the enzyme just needs to synthesize the bonds. Compare this with the blunt-end reaction, which requires pulling arbitrary blunt-ends together. So, blunt-end reactions are several times less efficient than sticky-end reactions. 3. Polynucleotide Kinases: These enzymes add phosphate group to the end of DN (or RN) chains, thereby setting the stage for ligase reactions. There is also an enzyme termed phosphomono-esterase (or phosphotase in short) that can remove the phosphate group. 4. Nucleases: Nucleases are enzymes that break the linking bonds between nucleotides to degrade the DN/RN chains. They come in different flavours. They could be exo or endo, depending on whether they remove nucleotides from the outside ends of a chain or degrade the insides of a chain. Then, there are nucleases specific for degrading DN or RN, or d.s DN, and 3 or 5 ends. Therefore, several combinations as depicted by (Exo/Endo) x (DN/RN) x (/d.s) x (3 /5 ) are possible. Examples of some are shown in Figure 3.3.

3 Lecture 3: January exonuclease polishes ends endonuclease removes interior d.s endonuclease cuts at random places Figure 3.3: Different flavours of nucleases dttp 5 T T T T T 3 dtp Figure 3.4: Terminal deoxynucleotidyl transferase converting blunt-ends to sticky-ends 5. Restriction Endonucleases: These are a class of nucleases that are important enough for separate listing. They cut DN chains at specific locations in a specific pattern. s a result, their end sequences can be either blunt-end or sticky-end (with 3 or 5 overhangs). Pho I is an example of a blunt-end cutter whose recognition site is GG/CC ( / indicates the point of cut). Bg II is another example, which recognizes a pattern at one point but cuts at some other point nearby. Its recognition site in terms of its two strands is (5 -GCCNNN/NNGGC-3, 3 -CGGNN/NNNCCG-5 ). From the recognition site, we see that Bg II is a sticky-end cutter with a 5 overhang. Information about all known restriction endonucleases can be found at the New England Biolabs website ( 6. Terminal deoxynucleotidyl transferase: This enzyme adds nucleotides to the blunt-ends of chains to make those ends sticky or dangling. It requires dntp molecules for synthesizing the nucleotides that are added. Figure 3.4 shows an example where a sticky-end is made from blunt-ends. 3.3 Labelling Labelling is the attachment of radioactive, flourescent or other markers to DN molecules. It is used to recognize a specific DN sequence amidst other DN fragments. n application could be identification of a certain DN sequence in the liver cell of a cancer patient. Suppose X is a 18bp DN sequence to be looked for, in a DN extract from the liver cell. Then we can do the following steps to test the presence of X. 1. Synthesize specific oligonucleotides: Synthesize the probe, which is the complementary oligonucletide sequence X. There are machines that take dntp molecules and synthesize them into a specific DN sequence. Figure 3.5 outlines the reactions involved for synthesizing C. The cost of synthesizing a 18bp sequence could range from $3 to $5 ($12 for purified sequence).

4 3-4 Lecture 3: January 28 C C Figure 3.5: Synthesis of a specific oligonucleotide sequence C ecor1 site 1100 bp circular DN ecor1 site agarose gel 8700 bp 8700 bp 1100 bp Figure 3.6: Illustration of Gel Electrophoresis 2. Hybridization: dd the probe X to the DN extract. If the sequence X were present, X will hybridize (base pair) with X resulting in a d.s region. This hybridization is thermodynamically favourable due to the complementary nature of the 18 bps. 3. Label: In order to recognize the hybridized d.s region, we need to add some markers. Previously radio-active P 31 was used, but nowadays more benign and safer flourescent tags are used. These are added to the probe before the probe is added to the DN extract. fter the hybridization reaction, excess markers are flushed out and the resulting mixture is observed for glows from the marker. These glows, if observed, would ascertain the presence of a d.s region involving X. 3.4 Separation DN being slightly negatively-charged will drift towards the positively-charged end, when put in a solution that is oppositely charged at either ends. It becomes interesting if the solution used is agarose gel which gives resistance to the passage of DN. Longer DN strands face more resistance from agarose than shorter ones. Thus, the latter drifts more towards the positive end than the former. This property can be used to separate out different-length DN strands and the process is called Gel Electrophoresis. Figure 3.6 shows an example of how a circular DN is cut into two pieces using ecor1 (a restriction endonuclease) and how the two pieces are separated using gel electrophoresis. There is another techique for separation called southern blot. Here the DN strands that are separated using electrophoresis are transferred ( blotted ) to a nylon membrane. This is done by pressing the nylon membrane over the gel (where the separated DN strands reside in different lanes), using a stack of paper towels and a weight on the top, and a buffer in the bottom. s the paper towels absorb the buffer, the DN

5 Lecture 3: January blots get transferred from the gel to the nylon membrane, from where they can be used later. n animation explaining southern blot can be found at similar process used for separating out RN is called northern blot. Western blot is a process for separating proteins. 3.5 Cloning Cloning is the process of amplifying a specific piece of DN after isolating it. One of the techniques for cloning involves a bacteriophage called λ. When λ virus is put in a E.Coli bacterial colony, the virus infects the bacteria and makes copies of themselves. The basic idea then is to insert the piece to be cloned at some innocuous place in the virus s DN (i.e., a place where the functioning of the virus is not affected). The manipulated virus is put in a E.Coli bacterial colony and 24 hours later, we get million copies of the virus and thus of the inserted DN piece. The process of insertion involves application of restriction endonucleases to the DN of λ virus (which is 50kbp long) to get sticky ends. The DN piece to be cloned can then be inserted into the virus s DN and the resulting solution pipetted out in minute amounts into E.Coli colony. The infection can be visualized as plaques, which are regions of clearing on the lawn of bacteria. While these viruses are parasites and kill the E.Coli bacteria, there are other organisms that can be used like plasmids which are symbiotic and thus allow the reuse of E.Coli bacteria. schematic picture of cloning DN in plasmids can be found at References [B99] Terence. Brown, Genomes, 1999.