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Share buttons are a little bit lower. Thank you! Please wait. Explain why projected financial statement analysis is a central strategy implementation tool. Explain how to evaluate the attractiveness of debt versus stock as a source of capital to implement strategies.Explain how management information systems can determine the success of strategy-implementation efforts. Explain business analytics and data mining.For example, in the credit card industry this could be signaled through a customer’s decline in spending on his or her card. This determination boils down to whether the post-retention profit generated from the customer is predicted to be greater than the cost incurred to retain the customer. Customers need to be managed as investments. For customers who are deemed “save-worthy,” it’s essential for the company to know which save tactics are most likely to be successful.Diagram a two-dimensional product-positioning map with specified criteria on each axis. Plot major competitors’ products or services in the resultant four-quadrant matrix.Look for vacant areas (niches). Develop a marketing plan to position the company’s products or services appropriately.Don’t serve two segments with the same strategy. Don’t position yourself in the middle of the map.Financial Forecasting and Planning Chapter 17. Chapter 14 Stock Analysis and Valuation. Copyright 2005 Prentice Hall Ch 8-2 Agenda Templates available in WebCT Mid-term will be after Spring Break. Due Feb 28 Templates available. Due MAR 3 at 2:00PM Templates. Successful strategy formulation does not guarantee. Successful strategy formulation does not guarantee successful strategy implementation. Less than 10 of strategies formulated. To make informed decisions about a company Helpful in managing the company Comparison. To use this website, you must agree to our Privacy Policy, including cookie policy. The book follows a modular approach and comprises learning outcomes, examples and activities. It is student-centered and the text is presented in a practical, clear and logical way. Overall, content treatment is coherent and current and relevant examples to the accounting profession are provided.No portion of this material may be reproduced, in any form or by any means, without permission in writing from the publisher. Continuing to use this website gives consent to cookies being used. For more information see our cookie policy. Answers Question Bank with answers Anna University BA7106 AM Notes. Syllabus 2 Marks with answers MBA 1st Semester - Regulation 2013 PG 1st. Semester Syllabus Notes BA7106 Notes Syllabus all 5 units notes Click below link to download the Notes and 2 marks with answers Question Bank We look forward to your suggestions and feedback. Usually, salt concentration and temperature are used to control the stringency. 1. Temperature of the reaction: High temperatures cause the DNA strands to move more rapidly. Imperfectly matched DNA hybrids will dissociate more rapidly at high temperatures. Therefore, higher temperatures favor the generation of perfectly matched hybrids. Obviously, if the temperature is too high, the two strands will not reanneal. 2. Salt concentration of the reaction mixture: High concentrations of salt allow imperfectly matched hybrids to be formed. At low salt concentrations, only perfectly matched hybrids will form. 3. Low salt and high temperature create high stringency conditions. The stringency of the match is controlled during the incubation of single stranded DNA (hybridization) or during the wash following the hybridization reaction. The temperature is the easiest parameter to adjust to obtain perfect matches. If using a short piece of single stranded DNA for hybridization, the final high stringency wash is often carried out 3-50C below the melting temperature to keep the perfectly matched strands hybridized but to eliminate imperfectly matched hybrids Luminex xMAP technology (SSO) Oligonucleotide probes are individually attached to up to 100 distinctly fluorescent microspheres, allowing the measurement of 100 different reactions in a single tube. This methodology is very appropriate for bulk processing of samples and it is used as our initial tissue-typing technique for all donors and patients. Detection of hybridization If the probe is labeled, hybridization can be detected. 1. Probes can be labeled using radioactive phosphate (P32) or by adding a modified or unusual nucleotide to a probe. The label is added to the probe either during or after synthesis of the probe 2. Binding can be detected by autoradiography (to detect P32) or through detection of color, chemiluminescence (light emitted by a chemical reaction) or fluorescence. Detectors may be X-ray film, ELISA plate readers, fluorescence or chemiluminescence detectors, or the human eye. DNA is negatively charged (due to the phosphate backbone) and moves away from the negative pole (cathode) and toward the positive pole (anode) in an electric field. Pieces of DNA are identified by size. Small pieces of DNA move more rapidly than large pieces of DNA. DNA is visualized on the gel by UV light if the DNA is stained with ethidium bromide or by X-ray film if the DNA is labeled with a radioactive isotope. Polyacrylamide gels are used to separate small fragments of DNA (5-500 base pairs) Agarose gels are used for DNA from 200 base pairs to 50,000 base pairs (50 kilobases). DNA Resolution The percent composition of acrylamide or agarose determines how easily pieces of DNA of different sizes can travel through the matrix; therefore, the percent composition of the gel will determine the range over which DNA fragments are resolved (separated from one another).Thermostable DNA polymerase (Taq DNA polymerase), E. coli DNA polymerase I, the Klenow fragment of that polymerase,and T4 and T7 polymerases (including Sequenase) require a DNA template. When starting with a mRNA template, the enzyme reverse transcriptase copies mRNA to make complementary DNA (cDNA). For example, a piece of DNA cut with the RE EcoRI into two fragments can be put together again by incubating with a ligase. Kinases add phosphates on to the 5' ends of DNA fragments. This enables DNA to be labeled with radioactive phosphate (P32) so that hybridization can be detected. Terminal transferase (TDT) adds nucleotides to the 3' ends of DNA molecules to make a single stranded tail. Terminal transferase can be used to create homopolymer tails (tails of all one nucleotide) for cloning or for labeling the DNA. Extraction of DNA from cells Any cell with a nucleus can be used as a source of DNA. Red blood cells do not contain nuclei; however, other cells in the blood like white blood cells are a good source of DNA. Cell lines like transformed B cells are also a good source of DNA. Because transformed cells can be grown in culture in the laboratory, they provide an inexhaustible supply of DNA. Many different protocols can be used to isolate DNA from cells. One protocol uses Triton X-100 (a detergent) to lyse the cell membrane releasing the nuclei. If the starting material was whole blood, these nuclei must then be washed extensively to wash away any hemoglobin released by the red blood cells. The heme portion of hemoglobin interfers with the gene amplification reaction used to determine HLA types. The nuclei are lysed using another detergent, Tween-20, and the DNA freed from the proteins bound to it by treatment with Proteinase K, an enzyme which destroys proteins. After the proteins are destroyed, the Proteinase K is also destroyed by incubation of the DNA at high temperatures (90oC). It is important to destroy the Proteinase K because it can degrade the enzyme used in the HLA typing reaction. Other methods may employ a solid phase to bind DNA for isolation. At least three different class I molecules are expressed on each cell of an individual. Each alpha (or heavy) chain can be divided into regions: three extracellular domains transmembrane region and cytoplasmic tail. Class I genes Some of the information specifying the amino acid sequences of the HLA class I molecules is located on human chromosome 6. The sequence of base pairs containing the genetic information that specifies a protein sequence defines a gene. The genes that encode the three class I alpha chains are located next to one another in the MHC.The DNA encoding the HLA molecule is copied (transcribed) into mRNA. After processing, the mRNA contains only the exon sequences. The mRNA is translated into protein by the ribosomes. The ribosome reads the genetic code to convert RNA sequences into a protein sequence. The genetic code consists of all possible triplet combinations of RNA bases. For example, the codon UCU (TCT in DNA) specifies the amino acid serine.These molecules are found on the surface of cells of the immune system like B cells and dendritic cells although other cell types may express these molecules under certain conditions (e.g., under the influence of cytokines). Class II genes The information specifying the amino acid sequences of the HLA class II molecules is located on human chromosome 6. The sequence of base pairs containing the genetic information that specifies a protein sequence defines a gene. These different gene sequences give rise to many different class I and class II allelic products (polypeptides).In the class II alleles, the second exons which encode the first domain of each polypeptide chain contain the majority of the allelic differences. Allelic differences can also be found in other exons. An example of alleles that do not differ at the protein sequence level is DRB1 110101 and DRB1 110102. Both alleles have the same protein sequence but differ in the codons used to specify that sequence (silent or synonymous substitutions). The W.H.O committee indicates silent changes by adding an additional two digits to the allele name. The HLA genes are codominantly expressed, that is, both copies encode proteins that are expressed on a single cell. Sometimes the two copies of chromosome 6, which carry the class I and II genes, exchange gene segments. If this happens in the germ cells (egg and sperm), that person's offspring may inherit the new combination.An expressed gene is one that encodes a polypeptide. The DR beta locus is called DRB1. Other copies of chromosome 6 carry two expressed DR beta genes. One locus is DRB1. The second beta chain locus is called DRB3 or DRB4 or DRB5. This sharing may result in difficulties in distinguishing specific HLA alleles. Class I loci are also highly polymorphic. In the class I alleles, the second and third exons which encode the first and second domains of the alpha chain contain the majority of the allelic differences. While null alleles are important to consider in matching of patient and donor, the importance of low vs.The C locus is called Cw so that it is not confused with the complement loci, which is part of the Class III. These molecules are less polymorphic than HLA-A, -B, and -C and are not considered important for HLA matching. HLA-E, HLA-G serology can not determine whether a donor and recipient who are typed as DR4 carry the same alleles of DR4. This is one reason that, in some situations such as typing for bone marrow transplantation, that serology has been replaced by DNA-based typing methods.For example, some DR7 haplotypes carry a DRB4 allele but it is not expressed as a DR53 molecule on the cell surface because of a defect in the gene (e.g., a termination codon halting protein synthesis). There are a number of ways of writing an HLA antigen. Using this method, we can generate millions of copies of a specific gene. PCR is a method of DNA synthesis A. DNA polymerase that is not destroyed when heated (thermostable): Taq polymerase. This enzyme comes from bacteria that live in hot springs. B. DNA template: Crude cell lysates containing heat denatured DNA can be used although better results are obtained with DNA that has been more extensively purified. C. Nucleotides. D. Primers Primers 1. One of the advantages of PCR is that it uses two primers so that both DNA strands are copied. The two single stranded primers must flank the region to be amplified. Since most of the polymorphism of the class II genes is found in the second exon, primers usually are designed to flank this region. Primers for class I alleles usually flank the polymorphism found in exons 2 and 3. 2. The primers hybridize to opposite strands of the DNA ladder. One primer is called a forward (or 5' or sense or coding) primer and the other primer is called a reverse (or 3' or antisense or noncoding) primer. 3. The primer sequences must complement (match) their target sequence and be sufficiently long (20-30 nucleotides) to bind to only the HLA gene that you want to amplify. 4. A primer should not contain any stretches of sequence that would anneal to the other primer (form primer dimers). For example, 5' AGCACTTTT and 5' TCCATAAAA would not be a good choice because the stretches of Ts and As would anneal. The polymerase would add complementary nucleotides to make a short double strand DNA called a primer dimer. DNA is amplified by using a three step procedure: A. DNA denaturation (94-96oC) to generate a single stranded template. These complementary single strands serve as templates for the production of new DNA. B. Annealing of the primers (45-65oC) using hybridization conditions that guarantee that the primers will bind to perfectly matched sequences (target sequence) and not to sequences that are not matched. The temperature of annealing controls, in part, the specificity of the amplification. The higher the temperature, the more specific the amplification until you get to the melting temperature of the primer. At that temperature, amplification does not work very well, if at all. C. Extension (replication) (synthesis of DNA) (around 72oC) is catalyzed by a DNA polymerase which brings the proper deoxynucleotide triphosphates (dNTPs) into position on the polymerizing chain. D. At the end of the first cycle, amount of target DNA is doubled E. The three steps are repeated over and over by simply changing the temperature of the reaction mix using an instrument called a thermal cycler. The newly synthesized strands serve as templates for synthesis in the next cycle (another advantage of PCR). Usually 25-30 cycles of amplification are carried out to yield millions of copies of the gene of interest. The biggest potential problem for PCR is contamination with previously amplified DNA. Failure to use the appropriate amplification conditions can cause amplification that will give either false positives (wrong alleles amplified) or false negatives (correct allele not amplified). Both probes and primers are single stranded pieces of synthetic DNA.The more differences between the matched and mismatched sequences, the easier it is to establish specific hybridization conditions. Probes are often called sequence specific oligonucleotide probes (SSOP). In SSO typing, the PCR amplification step generally involves the use of primers designed to amplify exons 2 and 3 (in the case of HLA Class I loci) or exon 2 (in the case of HLA Class II loci). The copies of the tissue-typing gene made by the PCR reaction are incubated with a panel of different oligonucleotide probes, with distinctive reactivities with different tissue-types. One strand of the DNA is often attached to a solid support (e.g., membrane or bead) to increase the speed of hybridization and to aid in the detection of a successful hybridization reaction. In either case, if the DNA contains a sequence that is exactly complimentary to the probe, the probe will anneal to the amplified DNA. Probes which are not exact complements of the amplified DNA are washed away during stringent washes. Use of PCR cycling parameters other than those recommended by the kit manufacturer may result in less efficient amplifications or give rise to non-specific reactions. Labeling of oligo probes to detect binding. Probes are usually end-labeled, tailed, or contain modified nucleotides. Labeling can be nonradioactive or radioactive (P32). If probes are attached to beads, the bead can be fluorescently tagged. Variations on the SSOP Forward SSO: amplified DNA is attached to a membrane and each membrane is hybridized with a different probe. Since multiple probes are required for even low resolution typing, numerous membranes are needed. In the standard dot blot format, multiple amplified DNAs are applied to each large membrane. All the DNAs are then simultaneously hybridized with each probe. Therefore, this dot blot format is much better suited to high volume batch testing. Reverse Dot Blot: The oligo probes (unlabeled) may be linked to the solid support and then hybridized to labeled, denatured, amplified DNA. Support can be a membrane, plastic plate or a bead. Labeled denatured amplified DNA is incubated with this chip and the binding of the DNA to individual oligos is detected by a excitation of the label with a laser. PCR-sequence specific primers (PCR-SSP) the PCR reaction is used to define whether the targeted tissue-typing gene is present or absent by using reagents in the PCR reaction specific for each of the variations seen in the tissue-typing genes. In this technique, PCR primers are designed to anneal only to a specific set of alleles or to a single allele. One or both primers include sequences unique to the allele(s). These unique sequences should be located at the 3' end of the primer for maximum specificity in the annealing step. Remember, that to get efficient amplification, both PCR primers must anneal to the DNA. The primer set can be designed to give an amplified fragment of a specific size which can detect its presence or absence by gel electrophoresis. If DNA for the appropriate HLA allele (or group of alleles) is added to the SSP mix, the primers will amplify the sequence of interest, and the amplified product can be visualized by gel electrophoresis. In tubes where the appropriate DNA required by the specific primers is not present, no HLA -specific amplification will occur and no HLA-specific product will be detected. The positive control primer pairs in each tube amplify segments of the human growth hormone gene. Competition for reagents occurs between the positive control and the typing primers. This may be seen as a dimming or even absence of the positive control band. Lanes exhibiting absence of the positive control band in the presence of an HLA typing band are acceptable.' Use of PCR cycling parameters other than those recommended by the kit manufacturer may result in less efficient amplifications or give rise to non-specific reactions. A modification of this approach is called ARMS (amplification refractory mutation system). PCR-sequencing or (SBT) Sequence Based Typing DNA sequencing can be used to determine the exact base sequence of the DNA or mRNA (cDNA) encoding an HLA molecule. The DNA sequence of the tissue-typing gene can be directly analysed by performing nucleotide sequence analysis of the PCR gene product. This method is used to achieve high resolution results for final stage testing of patients and donors and also to resolve any novel results. This is the ideal method for identifying the HLA alleles carried by an individual but is slower, more labor intensive, and more expensive than other typing methods. Following PCR amplification, the amplified DNA is cleaved with the restriction enzyme and the alleles are identified by the fragmentation pattern upon gel electrophoresis. Two problems with the technique are a failure to get complete cleavage which may make a homozygote look like a heterozygote and difficulty in finding appropriate restriction sites in all of the alleles at a locus. Denaturing gradient gel electrophoresis As amplified double stranded DNA is electrophoresed through a denaturing gradient gel, it reaches a point in the gradient where it denatures. This point is determined by the sequence of the DNA. Not widely used because the results may be difficult to interpret. Single stranded conformational polymorphism (SSCP). PCR-amplified DNA is denatured and electrophoresed on a polyacrylamide gel. Each single strand moves at a position related to its conformation as determined by its sequence. Not widely used because the results may be difficult to interpret, this method could be useful in comparing the alleles of two individuals. Heteroduplex formation Amplified DNA is denatured and allowed to reanneal under nonstringent conditions. If DNA strands are present which do not perfectly match (e.g., in a person heterozygous for the gene amplified), these will form heteroduplexes in addition to homoduplexes. These heteroduplexes will have an altered conformation compared to the homoduplexes and will migrate differently in an electric field. This method could be used to compare the alleles of two individuals or to determine an HLA type if compared to known alleles. Exonuclease-released fluorescence An sequence specific oligonucleotide labeled with reporter and quencher dyes is hybridized to target DNA. Addition of Taq polymerase and a locus-specific primer located 5' of the probe causes the reporter dye to be released during PCR. Once the reporter dye is separated from the quencher dye, fluorescence is produced indicating that the probe had hybridized to the DNA. Pyrosequencing. A modification of the sequencing reaction, this method adds only one nucleotide at a time. Addition to the growing chain is detected. This method is used for sequencing short segments of DNA and can potentially be used to determine which polymorphisms are linked together in a sequence.If the same sample is typed with the same set of probes each year, the interpretation of the hybridization results might change over time. Note that the hybridization result does not change; only the interpretation of that result.These cells are distinguished by unique polymorphisms, usually variability in the number of tandem repeated DNA sequences (i.e., short tandem repeats (STR) or variable number of tandem repeats (VNTR)) that are found in the donor versus the recipient. The concentration is critical: too little magnesium yields suboptimal amplification, too much is inhibitory.