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I_+Experiment+06+_+v+F10 - 6-1 Experiment 6 Examination of...

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6 - 1 Experiment 6 Examination of a Human Intron-less Gene of Lactate Dehydrogenase Introduction Examination of the primary amino acid sequence of bovine Lactate Dehydrogenase using the BLAST search program revealed the existence of a Human LDH gene without introns. The lack of introns in a eukaryotic gene is an unusual structural feature. Typically, eukaryotic genes coding for proteins contain introns that must be spliced out of primary transcripts generating functional mRNAs. Objectives 1. Amplify the Human intron-less LDH gene using PCR methodology. 2. Perform a restriction enzyme digest analysis on the PCR generated DNA fragment. 3. Examine the results for the expected size of the DNA fragment, for the expected restriction enzyme digestion patterns, and for the possibility of restriction enzyme polymorphisms that differ from the model sequence. Theory The determination of entire genomic sequences and the development of databases to store this information have allowed researchers interested in topics such as molecular evolution and human health to search these databases for useful information. Obtaining useful information from database searches is hypothesis driven, as with any good experiment. For researchers interested in human health, information on molecular evolution and human ancestry provides the material framework for comparing the sequences of specific genomic locations between similar groups of peoples who are less susceptible to a particular disease with those who may have high rates of a particular disease. The first completed human genomic sequence is haploid and of a male individual (why male?), thus it is not the representative sequence nor in any manner represents an “ideal” sequence for all humans, it merely serves as a reference sequence for which to base comparisons. Thus, acquiring additional human sequences from many members of the human family and comparing the sequences of genetic markers in the human genome is critical in order to make useful genetic distinctions between individuals and populations of individuals. Genetic markers are sequences such as repetitive DNA, specific chromosomal locations that are particularly susceptible to mutations, or protein coding genes. For protein coding genes, both the primary sequence of amino acids and the primary sequence of nucleic acids that code for the protein can be compared and used to examine genetic divergence between people. Open Reading Frames, Gene Families and Pseudo Genes The sequence information posted on databases and accessed for research interests must be considered carefully. Especially for biochemical research, protein sequence data can be complicated by the existence of open reading frames, gene families and pseudo genes. An open reading frame (ORF) is a segment of DNA that potentially codes for a protein with previously unknown identity and function.
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