Worksheets With Answers

Manila Science High School Taft Avenue, Manila 2nd Quarter Advanced Biology (Biotechnology) Worksheets Worksheet No. 1 DNA Cloning and Cloning Vectors 1. What are the steps involved in cloning a gene in a cell? There are several steps involved in cloning a gene. The specific methodology used in each step may vary depending on the type of DNA used, the host cell type and the ultimate goal of DNA cloning. The steps in gene cloning include: 1. 2. 3. 4. 5.

Isolation of DNA Ligating the DNA into a vector. Transformation of a host cell with the recombinant DNA (vector DNA with DNA insert) Selection of host cells harboring the recombinant DNA Screening of cells for those harboring the recombinant DNA or producing the appropriate protein product

2. What are the characteristics of a cloning vector? Often a foreign DNA is introduced into a host cell by inserting it into a cloning vehicle or vector that transports in into the host cell. Many of these vectors replicate independently in a host cell. A cloning vector must 1. Have an origin of replication so that the DNA can be replicated within a host cell 2. Be small enough to be isolated without underground degradation during purification 3. Have several unique restriction sites for cloning a DNA fragment so that the vector will be cut only once and several restriction sites for insertion will be available 4. Have selectable markers for determining whether the cloning vehicle has been transferred into cells and to indicate whether the foreign DNA has been inserted into the vector

3. Differentiate the 3 bacterial vectors. Vector Plasmids

Insert Size (1 kb equals 1000 nucleotides ≤ 10 kb

Bacteriophage

5-20 kb

Cosmids

35-45 kb

Characteristics Autonomously replicates in host cell, high or low copy number plasmids Packaged into protein coat, kills host cells, variable insert size Packaged like a bacteriophage, replicates like a plasmid without killing host cells

4. Define or discuss the following briefly a. multiple cloning site - also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites - a standard feature of engineered plasmids.

b. replica plating - technique in which one or more secondary Petri plates containing different solid (agar-based) selective growth media (lacking nutrients or containing chemical growth inhibitors such as antibiotics) are inoculated with the same colonies of microorganisms from a primary plate (or master dish), reproducing the original spatial pattern of colonies.

c. alpha complementation – two partial proteins can associate and form a functional beta-galactosidase

d. cohesive termini – Lambda phage vectors are derived from a 50-kb doublestrand genome that has single-strand complementary ends of 12 nucleotides called cohesive termini or cos. The cos ends (important for the lytic pathways) base pair forming a circular DNA molecule once the phage DNA is inside the host cell.

e. T-DNA – Transferred DNA is a special region in the plasmid, containing approximately eight genes that encode the disease characteristics, it is incorporated into the plant’s genome.

f. Ti plasmid – The most commonly used cloning vectors are plant viruses such as TMV, and the Ti, tumor inducing, plasmid of the soil bacterium Agrobacterium Tumefaciens. The bacterium’s large ti plasmid-greater than 200 kb-is what induces crown gall formation. Ti plasmid genes are involved in infection and induce plant cell division that leads to the tumorlike growth. 4. Give the characteristics of the following vectors for other organisms a. YAC’s – Yeast artificial chromosomes are useful for eukaryotic molecular studies. The yeast chromosome has the following necessary components: 1. A centromere distributes the chromosome to the daughter cells during cell division. 2. A telomere at the end of the yeast chromosome ensures that the end is correctly replicated and protects against degradation. 3. An autonomously replicating sequence consists of specific DNA sequences that enable the molecule to replicate YAC’s also have a gene that provides a way to detect an inserted DNA fragment. These components are joined to make a YAC that can replicate in yeast host cells.

b. BAC’s – Bacterial artificial chromosome are synthetic vectors and have been the most widely used DNA cloning systems for large genome sequencing projects. BAC’s are constructed using a very low copy of E. coli plasmid vector-the naturally occurring fertility factor plasmid-called the F factor. BAC vectors have been engineered to be approximately 74 kb. And contain cloning sites and selectable markers of different types. This type of vector is useful for analyzing large portions of complex genomes, whole genes, and constructing physical maps of genomic regions.

c. PCV – The properties of the Ti plasmid make it an efficient cloning vector in certain plants. The T-DNA which integrates into the plant’s chromosome can be used to transfer foreign genes into plants. For use in biotechnology, the engineered Ti plasmid lacks some of the genes that contribute to the cancerous properties. One way to transfer foreign DNA into all cells of a plant is to integrate the DNA of interest into few cells, which once transferred, divide and give rise to

the whole plant. Another way is to transfer DNA cells to the embryo by soaking seeds or buds of plant in a solution containing recombinant A.

d. MCV – (Mammalian Cell Vectors) Retroviruses are single-strand RNA viruses that show much promise for use as vectors in a wide variety of animal cells, including human. To replicate, the virus uses the enzyme reverse transcriptase to make a double-strand DNA molecule from RNA template. This DNA integrates stably into the chromosomes of dividing host cells, where transcription and translation of proteins occur. The adenovirus is a double-strand DNA virus. Like retrovirus, it can infect cells with high efficiency and has a broad host range. Moreover, unlike the retrovirus, adenovirus does not have to infect dividing cells. Worksheet no.2 Cell Transformation 1. When do you say that a cell is transformed? Exposing bacteria to a salt solution such as calcium chloride and applying a beat shock in the presence of DNA is usually sufficient for transferring DNA into cells, that is transformation. However, eukaryotes require more complicated methods. 2. When do you say a bacterium is competent? Some bacteria are naturally competent because they have the ability to take up extracellular DNA. However, many bacteria must be treated chemically to become competent. 3. How do cell walls of cells are broken down? What do you call a cell without a cell wall? In organisms that have cell walls, like fungi, algae and plants, enzymes are used to degrade the walls, producing protoplasts, or cells without walls. 4. Discuss the following methods: a. Electroporation – Protoplasts are exposed to a brief electrical pulse, which is thought to introduce transient openings in the cell membrane through which the DNA molecules enter. After transformation, cells are washed and cultured so that the cell wall re-forms and cell division begins.

b. Microinjection – To express a gene in all the cells of an animal, thus generating a transgenic organism, fertilized eggs or very early embryos are transformed by microinjection. DNA is injected directly into the nucleus of animal cells with an extremely fine pipette. After DNA is transferred into the cell, it is integrated into the chromosome and the transformed fertilized egg is implanted into an animal for the completion of development.

c. Biolistics – Very small microprojectiles made of gold or tungsten are coated with DNA and a shot at high velocity from a particle gun into cells or tissue. Because the projectiles penetrate the cell, the wall do not have to be removed.

d. Transfection – Transfection is the process of introducing nucleic acids into cells by non-viral methods

e. Transduction -. Transduction is the process by which DNA is transferred from one bacterium to another by a virus. Worksheet no.3 Constructing and Screening a DNA Library

1. What is a DNA library? its uses? Genes are sequences of DNA that code for the synthesis of RNA molecules used to make proteins. In order to study a gene, a researcher needs to isolate it from all the other genes in an organism’s DNA. One method involves the construction of a DNA library. It is a collection of DNA fragments that come from one organism and are stored in another organism, often a bacterium such as E.coli. 2. Steps in making a genomic library 1. First, total nuclear DNA is isolated from cells and cut with a specific restriction enzyme. 2. At the same time, a cloning vector is cut with the same restriction enzyme so that the vector is linearized and the ends are complementary to those of the genomic DNA fragments. 3. The two DNAs-genomic fragments and vector-are mixed in a test tube and DNA ligase is added to form recombinant molecules. 4. The recombinant DNA molecules are introduced into host cells, usually E. coli. Transformed bacterial cells, each containing recombinant plasmids or cosmids, multiply when plated onto antibiotic-containing medium. Each colony contains a specific soybean DNA fragment cloned into vector. Plaques, cleared areas where E. coli cells have lysed, contain recombinant DNA with genomic inserts. The collection of plaques or colonies that together contain all of the DNA fragments of a genome constitute the library. 3. Steps in making cDNA library 1. RNA from cells is isolated and used as a template to make DNA by a method called cDNA synthesis. 2. Reverse transcriptase catalyzes the reverse synthesis of cDNA from the mRNA template. The mRNA then is degraded with a ribonuclease or an alkaline solution, and DNA polymerase is used in the synthesis of the second DNA strand. 3. Double-strand DNA linkers with ends that are complementary to the cloning vector are added to the double-stranded DNA molecule before ligation into a cloning vector. 4. Recombinant clones are introduced to bacteria. Thus, the library is composed of cDNAs from the expressed mRNAs in cells. 4. Steps in screening libraries 1. The membranes are first treated so cells in colonies are lysed and cell debris are removed. 2. The remaining DNA is denatured to obtain single-strand molecules. 3. Single-strand DNA molecules bind tightly to the membrane by the sugar-phosphate backbones, and the unpaired nucleotides are free to base pair with a complementary DNA probe. 4. Hybridization of single-strand probes to these membranes is used to identify specific recombinant DNA molecules from the bacterial colonies or bacteriophage plaques transferred to the membranes. 5. Steps in expression libraries 1. E coli promoter is placed next to a unique restriction where DNA can be inserted 2. When a foreign gene or cDNA is cloned into an expression vector in the proper reading frame, the gene is transcribed and translated in the E. coli host cell. 3. When a library is made using an expression vector, only DNA-that is, genes (with introns if a eukaryotic gene) and cDNA (without genes)-inserted in-frame within the vector are expressed. 4. Antibodies are made by eukaryotic cells to response to proteins and other molecules that are recognized as proteins. A radioactively labeled antibody can be used to identify a specific protein made by one of the clones of the expression library. Antibody binding, a technique similar to nucleic acid hybridization, identifies the clone containing the gene expressing the specific protein. Worksheet 4

Reporter Genes 1. What is a reporter gene? Its uses? A reporter gene, connected to the DNA of interest and under the same control, is used to indicate whether the foreign gene or cDNA is being expressed. Reporter genes enable the investigator to quantify the level of expression, test the expression level from different promoters, and identify the tissue in which the gene is being expressed.

2. Examples of reporter genes and its sources. Many reporter genes are available that encode enzymes with readily assayed activities. 1. Green fluorescent protein (GFP) – This protein is produced by the jellyfish Victoria and interacts with a photoprotein acquorin, resulting in a green fluorescence. GFP naturally fluoresces green when the protein is excited with blue or ultraviolet light, and thus needs no substrate for the reaction to occur. Variants of GFP have been engineered to fluoresce at different wavelengths and intensities, although the signal is relatively weak, expression of GFP requires the use of very strong promoters or other regulatory sequences. A powerful application of GFP is the tracking of specific proteins, When the GFP gene is fused to a gene of interest to create a fusion protein. GFP fluorescence indicates the presence of the fused protein of interest, and the transport and fats of the fusion protein is followed by fluorescence.

2. Beta-glucoronidase gene (GUS) – Isolated from E. coli, this gene encodes an enzyme that catalyzes the breakdown of a variety of beta-D-glucuronides. In one type of essay, GUS generates a blue color that is formed by the breakdown of an intact, uncolored substrate. Alternatively, a more sensitive assay ca be used when a different substrate is used to generate a fluorescent product. The product can be quantified to measure the amount of product formed.

3. Chloramphenicol acetyltransferase (CAT) - is a bacterial enzyme that detoxifies the antibiotic chloramphenicol and is responsible for chloramphenicol resistance in bacteria. This enzyme covalently attaches an acetyl group from acetyl-CoA to chloramphenicol, which prevents chloramphenicol from binding to ribosomes.

4. β-galactosidase (lacZ) - is a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. Worksheet 5 Blotting techniques Complete the table below. Blotting technique 1. Southern Blotting

Proponents Edward Southern

2.Northern Blotting

James Alwine, David Kemp, and George Stark W. Neal Burnette

3. Western Blotting

Worksheet no.6 PCR 1. Uses of PCR.

Use/s Locating fragments Locating fragments Locating fragments

DNA

Probe used DNA probe

RNA

DNA/RNA probe

protein

antibody

Polymerase chain reaction allows a specific gene or other DNA region to be rapidly isolated from total DNA without the time-consuming task of screening a library. PCR is used to 1. Rapidly isolate specific sequences for further analysis or for cloning Identify specific genetic loci for diagnostic or medical purposes 2. Generate DNA fingerprints to determine genetic relationships to establish identity in forensics 3. Rapidly sequence DNA 2. Steps in PCR Initialization step: This step consists of heating the reaction to a temperature of 94-96°C (or 98°C if extremely thermostable polymerases are used), which is held for 1-9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR. Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94-98°C for 20-30 seconds. It causes melting of DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA. Annealing step: The reaction temperature is lowered to 50-65°C for 20-40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis. Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75-80°C. and commonly a temperature of 72°C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTP's that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases in one minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (also called geometric) amplification of the specific DNA fragment. 3. Requirements or materials needed for PCR PCR, as currently practiced, requires several basic components. These components are: • • • • •

DNA template, which contains the region of the DNA fragment to be amplified Two primers, which determine the beginning and end of the region to be amplified) DNA-Polymerase, which copies the region to be amplified Nucleotides, from which the DNA-Polymerase builds the new DNA Buffer, which provides a suitable chemical environment for the DNA Polymerase

4. Types of PCR RFLT PCR Allele specific PCR Assembly PCR Colony PCR Hot start PCR Nested PCR Touch down PCR RT PCR

Worksheet no.7 DNA sequencing 1. Uses of DNA sequencing DNA sequencing methods are valuable tools; they 1. Confirm the identity of genes isolated from DNA libraries by hybridization or amplified by polymerase chain reactor. 2. Determine the DNA sequence of promoters and other regulatory DNA elements that control expression. 3. Reveal the fine structure of genes and other DNA. 4. Confirm the DNA sequence of cDNA and other DNA synthesized in the test tube. 5. Help the scientists deduce the amino acid sequence of a gene or cDNA from the DNA sequence. 2. Discuss the method developed by Maxam and Gilbert The Maxam and Gilbert method of sequencing uses double-strand DNA molecules that are radioactively labeled at the 5’ end. The DNA is denatured and added to four tubes, one for each of the four nucleotides. To each tube is added a certain chemical that modifies and then removes a specific base from DNA strand. For example, a chemical is added to the C tube that removes cytosine. After the chemical reaction, the DNA backbone is cut where the base is missing. This process generates many radioactively labeled fragments of varying lengths, each ending where a specific base was eliminated. 3. Discuss the method developed by Sanger Dideoxy sequencing (also called chain- termination or Sanger method) uses an enzymatic procedure to synthesize DNA chains of varying lengths, stopping DNA replication at one of the four bases and then determining the resulting fragment lengths. Each sequencing reaction tube (T, C, G, and A) in the diagram contains • • • •

a DNA template, a primer sequence, and a DNA polymerase to initiate synthesis of a new strand of DNA at the point where the primer is hybridized to the template; the four deoxynucleotide triphosphates (dATP, dTTP, dCTP, and dGTP) to extend the DNA strand; one labeled deoxynucleotide triphosphate (using a radioactive element or dye); and one dideoxynucleotide triphosphate, which terminates the growing chain wherever it is incorporated. Tube A has didATP, tube C has didCTP, etc.

Worksheet no.8 Protein Methods 1. Discuss the following briefly: a. Protein gel electrophoresis – Electrophoresis is used to separate individual proteins by size or overall change. The proteins are separated by one or wodimensional polyacrylamide gel electrophoresis; polyacrylamide is used instead of agarose because it gives him a better resolution. Proteins are visualized in bands in a one-dimensional gel by a dye that binds to the proteins in the gel or by radiolabeling and autoradiography. First, the proteins are separated by charge in a slender tube gel in a process called isoelectric focusing. The tube is then placed horizontally along the top of a vertical slab gel, and proteins move by electrophoresis out of the tube and into the gel, which separates them by size. A detergent called sodium dodecyl sulfate (SDS) that is used in the second

dimension binds to the hydrophobic regions of proteins, giving the proteins an overall negative charge. Thus, in the second dimension, proteins are resolved by differences in size rather charge. Because all proteins are negatively charged, proteins move toward the positive end of the gel, with large molecules moving more slowly than smaller ones. Separating proteins in two dimensional achieves greater resolution of the molecules.

b. Protein engineering – Protein engineering is an exciting area that enables researchers to improve specific protein’s characteristics. By modifying the protein’s gene sequence, they can change protein structure to enhance protein function in specific ways. Changes can include increased stability such as resistance to degradation, PH change, temperature, oxidation and contamination; enhanced enzyme activity; a change in substrate specificity or enzyme activity in extreme or less-than-optimum conditions and increased nutritional value. The researcher must know the amino acid sequence, the chemical properties of the protein, the predicted three-dimensional structure, and in some cases the specific amino acids that contribute to the catalytic function of the enzyme. Several methods are available for changing the amino acids of proteins by mutating the corresponding nucleotides of the DNA. Frequently, only one nucleotide must be mutated to produce the desired amino acid change. One method, oligonucleotide-directed mutagenesis, uses a short single-strand DNA, an oligonucleotide 15 to 20 bases long, that is complementary to the region to be mutated except for the individual bases to be changed. The mutagenesis process is outlined in the following: 1. The base to be mutated must first be cloned. A viral vector, the bacteriophage M13, is used in the cloning process. M13 has both a single-strand DNA stage that is packaged within a viral coat and a double-strand phase, or replicative form (RF) that is synthesized after cell infection. The mutagenesis process is outlined in the following: 2, The oligonucleotide containing a specific change in sequence is then annealed to the single-strand circular DNA molecule, except at the specific mismatch site that cannot base pair. 3. The oligonucleotide serves as a primer for replication by DNA polymerase, and DNA ligase seals the ends of the newly synthesized circular DNA. 4. The DNA is transferred to E. coli where it is replicated. One strand of the original DNA molecule has the mutation from the oligonucleotide, and the other does not.Thus, some replicated RF double-strand molecules will have the mutation in their DNA sequence and others will not; the latter are called wild type. 5. Progeny phageare produced within the bacterial host cells and are released as a single-strand DNA packaged in a viral protein coat. 6. Mutant plaques are determined by hybridizing the oligonucleotide to the resulting plaques containing either wild-type or mutant DNA. Another way to mutate proteins is to substitute a DNA fragment, or cassette, containing the selected mutations for the same DNA fragment in organism. The DNA region containing the nucleotides to be changed is removed with a restriction enzyme and replaced with the cassette.This replacement is conducted in vitro a vector that is then transferred to E. coli for replication.

c. Protein sequencing – The linear of amino acids in a protein can be elucidated by a process called Edman degradation. In this method, the amino acids that make up the amino terminal end (the NH2 terminus) of the polypeptide strand are determined one at a time by chemical cleavage. The first amino acid is chemically modified to a phenylthiocarbamoyl amino acid and removed by acid treatment for identification. The next amino acid is now at the beginning of the chain (NH2 terminus) and the process is repeated until all the amino acids of the protein chain are identified.

Worksheet no.9 DNA Microarray Technology

1. Uses of DNA microarray technology One emerging technology is DNA microarray, which allows that analysis of a single genome in an entire experiment. DNA microarray is being used to 1. Analyze gene activity. Scientists are able to determine which genes are active. They are able to correlate changes in gene expression with changes in physiological or biochemical process such as photosynthesis in high and low light conditions. 2. Follow changes in genomic DNA. For example, changes in DNA occur in cancerous cells. Frequently, there are genomic modifications in the form of DNA amplification, point mutations, translocations, and deletions. Single nucleotide polymorphisms can be detected using microarrays that allow the detection of sequence variants in a specific gene. Microarrays are used to study gene expression in vivo. The genes that are transcripted at any one point in time are referred to as transcriptone. 2. Steps in DNA microarray technology The following outlines the steps in conducting DNA microarray analysis: 1. Isolating of DNA clones or preparing oligonucleotide for the microarray (probes). 2. Applying the cDNA or oligonucleotides to a substrate or chip. 3. Isolation of RNA-mRNA or total RNA. RNA is isolated from a particular cell or tissue state. 4. Preparation of the target-cDNA’s are synthesized from the RNA templates using the enzyme reverse transcriptase. 5. Hybridization of the fluorescently labeled-target sample to the DNA microarray. 6. Data acquisition and analysis. After hybridization, the DNA microarray is scanned with lasers to measure the amount of fluorescence in the hybridized target and probe. Worksheet no. 10 Applications of Recombinant DNA Technology 1.Give the use/s of Recombinant DNA Technology in the ff: a. Basic Biology - Answers questions about gene structure, gene function, and relatedness of genes and organisms b. Medicine - Answers questions about gene function

Makes human proteins for treating diseases such as diabetes, pituitary dwarfism, hemophilis Treats genetic diseases such as cystic fibrosis Produces more effective vaccines with fewer side effects Allows rapid, accurate diagnosis of infections and other diseases Better diagnosis, prevention and treatment of disease c. Industry - Improved production of antibiotics, amino acids, vitamins, and enzymes; also improved disposal of waste, including persistent toxic chemicals d. Agriculture - More rapid breeding of disease-resistant and improved plants More rapid development of superior breeds e. Criminal Investigation - Can determine if a biological sample such as blood, semen or tissue is from a particular person