The committee wants to see details of the PCR primers, the predicted PCR amplicon size and the expected size of the normal versus mutant DNA fragments after enzymatic digestion of the amplicon with Pvu II.- New Golden Gate Assembly simulation tool, either using existing Type IIS enzymes or through automated primer and overhang desig To obtain clearance to do the experiments, you must provide details of the protocols and the predicted outcomes of the analysis. This procedure involves the following separate steps in sequential order: Genomic DNA Isolation PCR of region of INs gene Pvu Il digestion of INS amplicon Visualisation of PCR controls and Pvu II digestion on 1% agarose gel As with all experiments involving humans, you need to obtain ethics clearance. You also plan to compare the patient DNA to the DNA of an individual known to be normal with respect to insulin secretion. You do not need to target the whole INS gene for amplification by PCR, only a region around the mutation and plan to PCR amplify an amplicon (region amplified) of 750 - 1000 bp. You decide to conduct a PCR-based analysis of the DNA of all other twelve patients to see if the same mutation occurs at their insulin locus. The base substitution also destroys the cleavage site for the restriction enzyme Pvu II (CAGCTG) - see separate document about restriction enzymes. ![]() FEATURES Location/Qualifiers Source 1.12565 /organism. ![]() TITLE Susceptibility to insulin dependent diabetes mellitus maps to a 4.1 kb segment of DNA spanning the insulin gene and associated VWR JOURNAL Nat. REFERENCE 1 (bases 1 to 12565) AUTHORS Lucassen, A.M., Julier,c., Beressi, J.P., Boitard, C., Froguel, P., Lathrop,M. SOURCE Homo sapiens human) ORGANISM Homo sapien Eukaryota: Metazoa: Chordata, Craniata Vertebrata Euteleostomi Mammalia Eutheria: Euarchontoglires Primates Haplorrhini Catarrhini Hominidae, Homo. end ACCESSION L15440 VERSION L15440.1 GI1307071 KEYWORDS insulin insulin-like growth factor 21 tyrosine hydroxylase. ![]() LQB280 Data sheet: Genbank accession #115440 LOCUS HUNINSTHIG 12565 bp DNA linear PRI 0 DEFINITION Homo sapiens tyrosine hydroxylase (TH) gene, 3' endi insulin (INS) gene, complete de insulin-like growth factor 2 (IGF2) gene, 5. 5584 - Base Name: Student # LQB280 Bioinformatics Exercise 3 Answer Template 1981 gaccgcagat tcangtgttt tittaagtas agtett gacct.googt cacagte 5041 secages et gestata gocce catcacoee ggagg09 gtgetect 5101 gotatoo goongaccee tgtcgocage ctcascag ctccatagte aggagatggg 5161 gagatgeta gogacaggee cougagaa gtactogat cacettes 9ctcccact 5221 gtgacactge cocggggge 99ganggagg toegacatat 99gcotto geetetaggt 5281 ocacacocag totoogtqac otecteta octogtoo accoggota gagatoote 5341 9gag gegadetagggetoe oogcagog cactgtet ctcotact gtgteeteet 5401 gtgtecetet gotecoge tattooggas octootete goggcacgte etageagtige 5461 yeagatega getgo999 ggcectotg cagcagoet geagocotty gooctogogg 5521 goteeetgea gaagetoge attatoase anteetgtae cageatege tootetace 5581 Bagus ctactcase tagaocage et cacaoag cccccacce googcetect 5641 gcacogaga agatata sagottga accagcocto ettgoogte tratatett 5701 9999 octo occasgoce cact tooge cactattoty agocotoce agetette 5761 aegetetete ootgoocaca 99gccasog cegoccagge coagoagoa gtggetto 5821 ccaaagoggc catgetatt gotgoetge tgococcace etgtggtoa wotecagta 5881 toggagette_99999tctct gagogocag qatagt999 focactgaga agtgaettet 5961 tottagtag ctetgacte ttggagtece cagagacett att aggas gegaat gaga 6001 acattecage aattttocce ccacctagcc ctcntt tatttttag agttatttet 6061 gatggagtoc ctatggagog aggaggeto getgagggag 999gtectge agggo99999 6121 getoggaagg tg99gagagg etgeegagag ccaccegeta tccccagate tgggcageco Oligonucletide Primer Sequence Tm (C) Location in genomic sequence 74 From base: 5041 Primers-cccacgctgcctgcctct999c9-3 To base: From base: 5891 Primers-CtCagagacccccgaagcrccca-3" 84 To base: SA03 Size of PCR fragment predicted: 963_ bp Recommended annealing temperature for PCR assay: 69 - 7L0 C) Predicted DNA fragment size(s) following digestion with Pvull: Normal (wild type) allele: bp.
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