DNA sequencing and Genomes Exam Questions and Answers Latest Update Graded A+

DNA sequencing and Genomes Exam Questions and Answers Latest Update Graded A+ Sanger sequencing development - Answers Developed in 1977 Fred Sanger •Called Sanger Sequencing - still used today Sanger sequencing - Answers DNA sequencing method based on selective incorporation of chain-terminating inhibitors (dideoxynucleotides) by DNA polymerase during in vitro DNA replication Dideoxynucleotides ddNTPs, and the Sanger sequencing reaction - Answers ddNTPs are chain terminating nucleotide bases - their 3'-hydroxyl (OH) group is replaced by a hydrogen molecule, so once added, the DNA polymerase is unable to add another nucleotide onto the strand of DNA. So once dideoxynucleotide is added it blocks further polymerisation by DNA polymerase •For Sanger sequencing, the ingredients are the same as for PCR, except the ratio of dNTP:ddNTP = 100:1 & a single primer is used. Only 1 cycle used. •The template can be a PCR product or plasmid. Sanger sequencing - s - Answers The reaction leaves strands of DNA of different lengths. To see the identity of the ddNTP at the end of the strand: •Size separation by gel electrophoresis •Used 4 separate sequencing reactions, each with a different radioactive ddNTPs (ddATP, ddGTP, ddCTP, ddTTP). •Electrophoresis using polyacralamide to separate by size •Use photographic paper to for detection of radioactive DNA strands •Manual process Sanger sequencing-1990's to- present - Answers Sequencing reaction using a mixture fluorescently labelled ddNTPs, and normal dNTPs. Each base has a different fluorescent "colour" Chain-termination PCR produces mixture of DNA strands of different lengths Size separation by gel electrophoresis, each DNA strand ends in a 'ddNTP terminator' Detection of fluorescent products using a laser (automated) - same method for microsatellite genotyping Genome - Answers The complete set of genetic information present in a cell or organism Genome size - Answers (the total number of the DNA base pairs in one copy of a haploid genome) varies widely across species Nuclear genome of the human genome - Answers DNA encoded within the 23 chromosome pairs found in nuclei Mitochondrial genome - Answers a small circular DNA molecule found within individual mitochondria encoding 37 genes (2 rRNAs, 22 tRNAs and 13 polypeptides) Human genome project - timeline - Answers •1977 - Sanger sequencing •1989 - UK Medical Research Council (MRC) & USA jointly funded the Human Genome Project •1990 - project begins: International team of researchers start - worlds largest collaborative biological project •2003 - Completed Human genome project - goals - Answers •Identify and map all genes in the human DNA from a physical and functional standpoint •Determine the sequences •Store information in databases •Discover efficient technologies for data analysis •Sequence other genomes of medical importance: mouse, yeast, Drosophila Human genome project - sequencing centre - Answers The Wellcome Trust Sanger Institute The sequence content of the human genome - Answers Transposons Transposons - Answers jumping genes Can replicate and insert into other parts of the genome •First discovered by Barbara McClintock, in maize •Two categories: ● Retrotransposons (transpose via mRNA) ● DNA transposons •Most mobile elements in bacteria - Answers DNA transposons Most mobile elements in eukaryotes - Answers retrotransposons (e.g. LINEs), but eukaryotic DNA transposons also occur Retrotransposons - Answers encode reverse transcriptase. •After transcription, the RNA is converted into DNA, then inserted into another part of the genome. •Alu elements comprise 11% of the human genome. •Alu is a SINE. These do not encode a functional reverse transcriptase protein and exploit LINE transposition components to transpose Simple sequence repeats - Answers Microsatellites and Minisatellites