GENETICS ENGINEERING RESUME

By : Niswati Zahro ( 100210103068)

BIOLOGY EDUCATION STUDY PROGRAM DEPARTEMENT OF MATEMATHIC AND SCIENCE EDUCATION FACULTY OF TEACHER TRAINING AND EDUCATION JEMBER UNIVERSITY 2011

A. DEFINITION OF GENETICS ENGINEERING According to Nurchalis in Zamroni (2007), genetics engineering was a human effort in the science of biology by manipulating (engineering) cells, or genes contained in a particular organism with the aim of producing is new types of organisms are genetics identically. Genetics engineering or recombinant DNA technology is a set of methods used to locate, analyze, alter, study, and recombine DNA sequences. It is used to probe the structure and function of genes, address questions in many areas of biology, create commercial products, and diagnose and treat diseases. The term recombinant is used because frequently the goal is to combine DNA from two distinct sources. Genes from two different bacteria might be joined, for example, or a human gene might be inserted into a viral chromosome. Recombination is the exchange of genetic information between DNA molecules that was causing to increases genetic variation.

B. RECOMBINANT DNA TECHNIQUES 1. Cutting and Joining DNA Fragments Restriction enzymes (also called restriction endonucleases) that recognize and make doublestranded cuts in the sugarphosphate backbone of DNA molecules at specific nucleotide sequences. These enzymes are produced naturally by bacteria, where they are used in defense against viruses. Three types of restriction enzymes have been isolated from bacteria. Type I restriction enzymes recognize specific sequences in the DNA but cut the DNA at random sites that may be some distance (1000 bp or more) from the recognition sequence. Type III restriction enzymes recognize specific sequences and cut the DNA at nearby sites, usually about 25 bp away. Type II restriction enzymes recognize specific sequences and cut the DNA within the recognition sequence. More than 800 different restriction enzymes that recognize and cut DNA at more than 100 different sequences have been isolated from bacteria. Restriction enzymes are the workhorses of recombinant DNA technology and are used whenever DNA fragments must be cut or joined. There are two types fragment end produce of cutting DNA fragment by restriction enzyme i.e : a. Cohessive ends or sticky ends, because they are complementary to each other and can spontaneously pair to connect the fragments. For example HindIII cuts the sugarphosphate backbone of each strand at the point indicated by the arrow, generating fragments with short, single-stranded overhanging ends: 5-A AGCTT-3 3-TTCGA A-5

b. Blunt-ended fragments 5-AAG 3-TTC CTT-3 GAA-5

After the DNA fragment was cut by restriction enzyme, DNA fragments can be joined together permanently by the enzyme DNA ligase, which seals nicks between the sugar-phosphate groups of the fragments.

Figure 1. Restriction enzymes make double-stranded cuts in the sugar phosphate backbone of DNA, producing cohesive, or sticky, ends.

2. Viewing DNA Fragments Gel electrophoresis is used to separate DNA molecules based on the sizes of the resulting DNA fragments. Small DNA fragments migrate more rapidly than do large ones passed through the gel to the positive end of the gel when an electricalcurrent is passed through the gel because the phosphate of each DNA nucleotide carries a negative charge. Then, DNA fragments of known length (a marker sample) are placed in another well. By comparing the migration distance of the unknown fragments with the distance traveled by the marker fragments, one can determine the approximate size of the unknown fragments.

Figure 2. Gel electrophoresis can be used to separate DNA molecules on the basis of their size and electrical charge

The DNA fragments are still too small to see. There are several ways to visualization it i.e: a. The simplest procedure is to stain the gel with a dye specific for nucleic acids, such as ethidium bromide, which wedges itself tightly (intercalates) between the bases of DNA.When exposed to UV light, ethidium bromide fluoresces bright orange; so copies of

each DNA fragment appear as a brilliant orange band The original concentrated sample of purified DNA contained millions of copies of a DNA molecule, and thus each band represents millions of copies of identical DNA fragments. b. Visualization by adding a radioactive or chemical label to the DNA before it is placed in the gel. Nucleotides with radioactively labeled phosphate (32P) can be used as the substrate for DNA synthesis and will be incorporated into the newly synthesized DNA strand. c. End labeling, the bacteriophage enzyme polynucleotide kinase is used to transfer a single 32P to the 5_ end of each DNA strand. Radioactively labeled DNA can be detected with a technique called autoradiography, in which a piece of X-ray film is placed on top of the gel. Radiation from the labeled DNA exposes the film. Chemical labels can be detected by adding antibodies or other substances that carry a dye and will attach to the relevant DNA, which can be visualized directly.

Gel electrophoresis is used widely in recombinant DNA technology; it is often employed when there is a need to determine the number or size of DNA fragments or to isolate DNA fragments by size.

3. Locating DNA Fragments with Southern Blotting and Probes One approach to locate the desired fragments in such a large pool of DNA is to use a probe, which is a DNA or RNA molecule with a base sequence complementary to a sequence in the gene of interest. The bases on a probe will pair only with the bases on a complementary sequence and, if suitably tagged with an identifying label, the probe can be used to locate a specific gene or other DNA sequence. Steps to use a probe are: cuts the DNA into fragments by using one or more restriction enzymes separates the fragments with gel electrophoresis. The separated fragments must be denatured and transferred to a thinner solid medium (such as nitrocellulose or nylon membrane) to prevent diffusion. There are techniques to transfer the denatured, i.e : a. Southern blotting is used to transfer the denatured single-stranded DNA fragments from a gel to a thin solid medium or membrane that was placed in a hybridization solution of a radioactively or chemically labeled probe (see Figure 18.5). There are two result that are:

o the bound probe i.e. DNA fragments on the membrane that bear complementary sequences; bound probe is detected by autoradiography or another method for chemically labeled probes. o unbound probe that will remove by washing the membrane; b. Northern blotting is used to transfer RNA from a gel to a solid support. The hybridization of a probe can reveal the size of a particular mRNA molecule, its relative abundance, or the tissues in which the mRNA is transcribed. Here, the probe is usually an antibody, used to determine the size of a particular protein and the pattern of the proteins expression. c. Western blotting is the transfer of protein from a gel to a membrane.

Figure 3. Southern blotting and hybridization with probes can be used to locate a few specific fragments in a large pool of DNA.

4. Cloning Genes Gene cloning is procedure to obtain copies of a specific DNA fragment by using a bacterial cell and allow the cell to replicate the DNA fragment. Gene cloning must has vector to run its process, that was called cloning vectors.

Characteristic of cloning vectors: stable, replicating DNA molecule to which a foreign DNA fragment can be attached for introduction into a cell an origin of replication, which ensures that the vector is replicated within the cell selectable markers, which enable any cells containing the vector to be selected or identified one or more unique restriction sites into which a DNA fragment can be inserted.

There are three types of cloning vectors genes i.e. :

a. Plasmid vectors

Plasmids are circular DNA molecules that was able to replicate independently of the chromosome in bacteria. The way is DNA and the plasmid are cut by the same restriction enzyme to result the sticky ends on the foreign DNA and the plasmid so, the DNA and plasmid can mixed together by using DNA ligase in the sugarphosphate backbone, creating a recombinant plasmid that contains the foreign DNA fragment. There are some method, that is: restriction cloning, on the restriction cloning, the sticky ends of the plasmid are complementary to each other; so the two ends of the plasmid will often simply reanneal tailing, the single-stranded ends of the plasmid are complementary only to the singlestranded ends of the foreign DNA. to use the enzyme T4 ligase, which is capable of connecting any two pieces of blunt-ended DNA. linkers to add complementary ends to DNA molecules. Linkers are small, synthetic DNA fragments that contain one or more restriction sites. A particular restriction site can be added at almost any desired location; so any two pieces of DNA can be cut and joined. Transformation is an introducing the plasmid into bacterial cells so bacterial cells can take up DNA from the external environment and then the plasmids replicate and multiply. Transformation can run naturally, treated chemically or physically.
b. Bacteriophage vectors

Bacteriophage vector is bacteriophage which infects E. coli. The essential genes of the phage _ genome are located in a cluster. Strains of phage called replacement vectors. The nonessential genes can be removed by using EcoRI. Foreign DNA cut with EcoRI will have sticky ends that are complementary to those on the ends of the essential genes, to which it can be connected by

ligase. The chromosome possesses short, single-stranded ends called cos sites that are required for packaging DNA into a phage head. The recombinant phage chromosomes can then be packaged into protein coats and added to E. coli. The phages inject their recombinant DNA into the cell, where it will be replicated. Only DNA fragments of the proper size and containing essential genes will be packaged into the phage coats, providing an automatic selection system for recombinant vectors.
c. Cosmid vectors

Cosmids are small plasmids that carry phage _ cos sites; they can be packaged into viral coats and transferred to bacteria by viral infection. Because all viral genes except the cos sites are missing, a cosmid can carry more than twice as much foreign DNA as can a phage vector. .

To ensure transcription and translation, a foreign gene is usually inserted into an expression vector. These additional sequences may include: 1. A bacterial promoter, to allow transcription of the foreign sequence to be regulated by adding substances that induce the promoter. 2. A DNA sequence that, when transcribed into RNA produces a prokaryotic ribosome binding site. 3. Prokaryotic transcription initiation and termination sequences. 4. Sequences that control transcription initiation, such as regulator genes and operators.

The DNA sequence to be cloned in the first place can find between millions or billions of base pairs of DNA in a cell by using shotgun cloning. One first clones a large number of DNA fragments, knowing that one or more contains the DNA of interest, and then searches for the fragment of interest among the clones. A collection of clones containing all the DNA fragments from one source is called a DNA library. There are two kinds of DNA library i.e. : a genomic library, it is created by cutting genomic DNA into overlapping fragments and cloning each fragment in a separate bacterial cell A cDNA library is created from mRNA that is converted into cDNA and cloned in bacteria.

5. Polymerase Chain Reaction (PCR) The polymerase chain reaction is a technique that allows DNA fragments, even a single molecule, to be amplified a billionfold within just a few hours. The process can be followed by this figure.

Figure 4. The polymerase chain reaction is used to amplify even very small samples of DNA. The polymerase chain reaction is an enzymatic, use the dubbed Taq polymerase that was isolated from Thermus aquaticus, is remarkably stable at high temperatures and is not denatured during the strand-separation step of PCR; so it can be added to the reaction mixture at the beginning of the PCR process and will continue to function through many cycles, in vitro method for rapidly amplifying DNA. In this process, DNA is heated to separate the two strands, short primers attach to the target DNA, and DNA polymerase synthesizes new DNA strands from the primers. Each cycle of PCR doubles the amount of DNA.

6. Analyzing DNA Sequences a. DNA sequencing The determination of the sequence of bases in DNA. Sequencing allows the genetic information in DNA to be read, providing an enormous amount of information about gene structure and function. b. In situ hybridization Process to determine the chromosomal location of a gene or the cellular location of an mRNA, to determine the tissue distribution of specific mRNA molecules, serving as a source of insight into how gene expression differs among cell types. The name is derived from the fact that DNA or RNA is visualized while it is in the cell (in situ). c. DNA footprinting A technique to determine which DNA sequences are bound by such proteins. When the protein is absent, cleavage is random along the DNA, producing a continuous ladder of

bands on the autoradiograph. When the protein is present, it binds to specific nucleotides and protects their phosphodiester bonds from cleavage. The omission of cleavage in the area protected by the protein and label of fragments terminating in the binding site appear on the autoradiograph cause the forming a gap, or footprint, on the ladder of and the position of the footprint identifies those nucleotides bound tightly by the protein. d. Microarray are the microscopic array of single-stranded DNA molecules immobilized on a Solid surface by biochemical synthesis that called as DNA chips, gene chips, biochips, DNA microarrays or simply the arrays.

Figure 5. process of Microarray

e. Enzym Linked Immunosorbent Assays (ELISA) ELISA (Enzyme Linked Immunosorbent Assay) is biochemical method in laboratory analysis and diagnostics such as peptides, proteins, antibodies and hormones can be detected

selectively and quantified in low concentrations among a multitude of other substances. Although many variants of ELISA have been developed and used in different situations, they all depend on the same basic elements: Coating/Capture: direct or indirect immobilization of antigens to the surface of polystyrene microplate wells. Plate Blocking: addition of irrelevant protein or other molecule to cover all unsaturated surface-binding sites of the microplate wells. Probing/Detection: incubation with antigen-specific antibodies that affinity-bind to the antigens.

Signal Measurement: detection of the signal generated via the direct or secondary tag on the specific antibody.

Figure 6. Diagram of popular ELISA formats. 7. Cloning Cloning is typically done by taking a single skin cell from the animal to be cloned. That cell contains DNA in its nucleus with complete blueprints of the entire animal. An egg is then taken from some other animal, and the nucleus is removed from that egg and discarded. The remainder of the egg is combined with the skin cell. The result is a single cell that acts as if it were a fertilized egg. It can be implanted in a surrogate mother and eventually be born.

Figure 7 Cloning of Dolly step by step. 1: Skin cells removed from the udder of a white faced Finn Dorset Ewe and placed in a growth culture containing very low nutrients: the cells are starved, stop dividing and the active genes are switched off. 2: The DNA is removed from an egg cell from a Scottish Blackface Ewe leaving the egg cell cytoplasm containing mitochondria. 3: Theskin cell and the egg cell are placed next to each other and subjected to an electric pulse to cause fusion. The fused cell contains 46 chromosomes from the Finn Dorset Ewe in the cytoplasm of the Scottish Blackface Ewe. The fused cell is given a 2nd electric pulse to activate cell division and turn on the genes necessary for growth of the embryo. 4: The embryo is then implanted into the uterus of a different Scottish Blackface Ewe. Dolly, a cloned white faced Finn Dorset lamb, is born 8. Hybridoma And Antibody Monoclonal Hybridomas are cells that have been engineered to produce a desired antibody in large amounts, to produce monoclonal antibodies. Monoclonal antibodies are used, for instance, to distinguish subsets of B cells and T cells.

Figure 8: hybridoma technique to produce antibody monoclonal To produce monoclonal antibodies, B cells are removed from the spleen of an animal that has been challenged with the relevant antigen. These B-cells are then fused with myeloma tumor cells that can grow indefinitely in culture (myeloma is a B-cell cancer). This fusion is performed by making the cell membranes more permeable. The fused hybrid cells (called hybridomas), being cancer cells, will multiply rapidly and indefinitely and will produce large amounts of the desired antibodies.

9. Transgenic Organism a. Animal transgenic A transgenic animal is an animal in which foreign DNA has been incorporated into its original DNA. Transgenic animals are altered so that their DNA produces chemicals that normally they would not produce. Transgenic animals can be divided into five major categories: Disease models are animals that have been modified to exhibit the symptoms and progression of a particular disease, so that treatments for that disease can be tested on them. Transpharmers are animals modified to express a particular protein or suite of proteins in their milk to avoid animal sacrifice when obtaining the drug. The proteins can be purified to produce medicines and hormones to treat humans, or can possibly be administered as medicinal milk itself. Xenoplanters are animals that have been engineered to not express the foreign antigens that normally prevent the transplantation of their organs into humans. Food sources are animals that grow bigger or faster to produce more food in a shorter amount of time with fewer resources. Scientific models are animals producing more or less of a particular protein than usual, letting us observe that proteins purpose in biological mechanisms or development, which can in turn be

Figure 9 : The World's First Transgenic Monkey, ANDi.(Bradley: 2006)

b. Transgenic plants Transgenic plants are plants that have been genetically modified by inserting genes directly into a single plant cell. Transgenic crop plants modified for improved flavor, pest resistance, or some other useful property are being used increasingly. Transgenic plants are unique in that they develop from only one plant cell.

Figure 10 : A "normal" melon (left) after five days, and a transgenic melon (right) after fifteen days. The ability of the modified fruit to be edible for a much longer period is a trait desirable for those in the produce business.

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W. H. Freeman Pierce, Benjamin. 2005. Genetics: A Conceptual Approach, 2nd Ed. New York: W. H. Freeman And Company, 515. Bible, A. Valey. 2002. Cloning, Genetic Engineering And Ivf. Bradley, M. & Brosius, J. 2006. Transgenic Animal. Worcester Polytechnic Institute Genetics Education. 2007. Cloning and Stem Cells. Australia: The Australian Genetics Resource Book. Pandey, S. 2010. Hybridoma Technology For Production Of Monoclonal Antibodies. Rbpm: 1 (2) : 88-94. Parsad, Rajender. Design And Analysis Of Microarray Experiment: Some Concepts. New Delhi: Lasri , 6 (1): 85-100. Pena, Leandro. 2004. Transgenic Plants: Methods and Protocols.New Jersey: Humana Press. Pierce, Benjamin A. 2005. Genetics A Conceptual Approach. New York: W. H. Freeman And Company Pierce. 2011. Elisa Technical Guide And Protocol. Usa: Thermo Scientific. Xu, Wayne. 2003. Introduction To Microarray Technology.Minnesota: Cgc Institute.

Zamroni. 2007. Controversion Of Cloning Into Human. MAZAHIB, 6 (1): 25-35.