Genetics Dideoxy Sequencing Notes Part 2

-The incubation times depend on the size of the DNA being copied. -After 25-30 cycles, there is a final extension step at 68 °C to ensure the polymerase completes the extension of all DNA strands. -After one PCR cycle, the dsDNA template will be denatured into two ssDNA molecules. Then, the polymerase will fill in the complementary strand to form two dsDNA copies. Using the same logic how many dsDNA copies are present after two PCR cycles? Polymerase chain reaction (PCR) is a technique used to amplify (copy) DNA. Suppose a single, linear molecule of double-stranded DNA (dsDNA) is amplified by PCR. After 30 PCR cycles (a typical number of cycles), how many molecules of dsDNA will there be? Consider that a typical PCR does not start with a single molecule of template DNA, but rather something in the range of 25 nmol of template DNA. What does this tell you about the potential of PCR to amplify DNA? Answer: - ~60 billion -PCR is an efficient technique with the potential to produce a large amount of DNA Place the steps of generating a gene-knockout mouse in order from the disruption of the target gene to generating homozygous knockouts. Suppose in one experiment, the stem cells used are from a black-coated mouse, and the early mouse embryo used is a white-coated embryo. Disrupt target gene by inserting neo+ and link tk+ to disrupted gene -> Breed mice to generate homozygous knockouts Answer: 1) transfer disrupted gene into stem cells from a black mouse 2) allow recombination with genome to generate neo+ tk- cells 3) select for neo+ and select against tk+ to isolate cells from homologous recombination 4) inject neo+ cells into white early embryo and implant into surrogate 5) identify chimeric progeny with target-gene knockout cells Explanation: -Genetic manipulation in cell culture is required before a knockout mouse can be generated. How are different mouse strains valuable in studies of human disease? Answer: -Distinct strains of mice have bee created as models of specific human diseases Explanation: -Laboratory mice have been carefully bred to create groups of mice with specific traits that are useful in studies of human disease. Which of the following is an example of gene therapy? Answer: -Replacing a disease-causing allele for a gene with a wild-type allele Explanation: -Mutations in a particular gene can result in the absence of a functional protein and cause disease in affected individuals. Gene therapy can be used to restore the function of a mutated gene, so that the affected individual can produce the correct protein, and therefore cure, or lessen the severity of the disease. Which of the statements defines somatic gene therapy? Answer: -The transfer of recombinant DNA in people with disease to cure or decrease the symptoms of the disease Explanation: -Think about the possible therapeutic solutions for individuals affected with a genetic disease. Consider that integrating new DNA into an individual's cells can counteract mutations and change their phenotype. A biotechnology firm wants to produce a strain of giant Drosophila so that genetics students do not strain their eyes looking at tiny flies. A hormone called shorty substance P (SSP) normally inhibits growth in the flies. The enzyme runtase synthesizes SSP from the compound XSP in a single-step catalytic reaction. A researcher isolated and sequenced runtase cDNA, but the researcher does not know the location of the runtase gene in the Drosophila genome. Initial attempts to delete, inactivate, or otherwise mutate this gene failed. Therefore, the researcher tries gene augmentation, or adding new genes to a cell. Which gene augmentation method could produce giant flies? Answer: -insert a gene that expresses dsRNA for runtase, which would invoke the RNAi pathway and shut down runtase expression Explanation: -Increased runtase expression causes greater SSP production and diminished fly growth. Runtase enzymatically produces the growth-suppressing SSP hormone from the compound XSP. Thus, SSP itself cannot be genetically modified, because no gene encodes SSP. You discovered a mouse gene with an unknown function. You do not know the location or sequence of this gene in the mice genome, but a similar gene has been isolated and sequenced in yeast. How might you determine whether this gene is essential for development in mice? Answer: -Create a targeting vector using a cloned copy of the gene to develop a knockout mouse. If the gene is essential for development, no offspring homozygous for knockout will be born. Explanation: -You can suppress a gene and determine how the phenotype changes to determine a gene's function in vivo. How do the goals of the HapMap project differ from the goals of the Human Genome Project? Answer: -The HapMap Project identifies common genetic variations among individuals, whereas the Human Genome Project sequenced the entire human genome Explanation: -The Human Genome Project was completed before the HapMap Project. The information gained from the Human Genome Project is being used to identify potential targets in the HapMap Project. Most scientists consider the Human Genome Project (HGP) to be the most significant scientific project of the 21st century. Choose the statements that describe the key findings of the Human Genome Project. Answer: -The human genome contains approximately 25000 genes. -There are approximately three billion base pairs in the human genome. Explanation: -Start by eliminating discoveries made prior to the completion of the first draft of the human genome sequence. The genome of Drosophila melanogaster, a fruit fly, was sequenced in 2000. However, this sequence consisted mainly of euchromatin. The heterochromatin was not sequenced until 2007. Most completed genome sequences do not include heterochromatin. Why is heterochromatin usually not sequenced in genomic projects? Answer: -Heterochromatin contains many short repeated sequences, which makes it difficult to assemble heterochromatin sequences into large contigs. Explanation: -Next-generation sequencing technology uses a shotgun approach that breaks large genomic DNA into short segments for easier sequencing. Unique overlapping regions of these short segments are used to computationally reassemble the pieces into long stretches of DNA. -Nuclear DNA comes in two forms: euchromatin and heterochromatin -Euchromatin is lightly packed and contains actively transcribed genes. -Heterochromatin is densely packed, and genes located in heterochromatic regions are largely silenced Whole-genome shotgun sequencing is a method used to sequence genomic DNA. Arrange the steps of whole-genome shotgun sequencing in order from first to last. First step -> Last step Answer: 1) Isolate genomic DNA 2) Break DNA into large fragments that are approximately 150,000 base pairs in length 3) Insert DNA into BACs 4) Break DNA into fragments that are approximately 1000 base pairs in length 5) Insert DNA into plasmids 6) Sequence DNA 7) Computationally assemble sequences from each BAC 8) Computationally assemble the genome from overlapping BACs Explanation: -Whole-genome shotgun sequencing requires generating bacterial artificial chromosomes (BACs), which allow bacteria to maintain and replicate very large fragments of foreign DNA. After BACs are generated, plasmids are used to amplify smaller fragments of foreign DNA. Whole-genome shotgun sequencing works by reconstructing the genome from small, overlapping pieces. With current sequencing technology, contigs are integral to understanding the entire genome of an organism. What is a contig? Answer: -a continuous DNA sequence constructed from overlapping DNA fragments Explanation: -The term contig comes from the word contiguous which means connected without a break. Recent advances in genome sequencing technology facilitate whole-genome sequencing of non-model organisms. Select the scientific questions that a geneticist can answer with the whole-genome sequence of a single individual of a non-model organism. Answer: -What are adaptive and neutral loci in an organism? -How are organisms related? Different types of environmental studies are listed. Select the metagenomic studies. Answer: -DNA of primate gut contents is sequenced to determine microbial diversity in their food source. -Genetic analyses of deep sea sediment samples characterize regional microbial composition. Explanation: -Metagenomics is also sometimes referred to as environmental genomics. Suppose a researcher studied gut tissue samples from patients with Crohn's disease, an inflammatory gut disorder, and normal controls. He found that the identity and relative abundance of gut bacterial species varied within both the Crohn's and the control populations. He also discovered general differences between the two populations. The guts of Crohn's patients had reduced levels and diversity of bacteria from the phylum Firmicutes compared to those of controls. The guts of Crohn's patients also had higher levels of bacteria from the phylum Proteobacteria than those of controls. Which type of study did the researcher perform? Which conclusions might you be able to draw from the data? Answer: -Metagenomic -Two normal individuals may have different compositions of bacteria in their guts -Bacteria from the phylum Firmicutes may provide protection against Crohn's disease Explanation: -In modern biology, the suffix -omic refers to studies of all the components of a particular set. Consider if the research was studying the gut genes, mRNA, proteins, or microfauna in the Crohn's patients. James Noonan and his colleagues (2005. Science 309:597-599) set out to study genome sequences of an extinct species of cave bear. They extracted DNA from 40000-year-old bones from a cave bear and used a metagenomic approach to isolate, identify, and sequence the cave bear DNA. Why did they use a metagenomic approach when their objective was to sequence the genome of one species, the cave bear? Answer: -The cave bear samples could be heavily contaminated so scientists used metagenomics to sequence all of the DNA. They then compared the sequences obtained with modern bear DNA to idenitfy which DNA frafments were from the cave bear Explanation: -Metagenomics sequences all the DNA in an enivornmental sample without purifying the sample of interest. A linear piece of DNA was broken into random, overlapping fragments and each fragment was then sequenced. The sequence of each fragment is shown here, listed in random order. 5 −ATCAAAAGC−3 ′ ′ 5 −TCAAAAGCATAGAGGTACC−3 ′ ′ 5 −GAGGTACCCTTC−3 ′ ′ 5 −CTATCAAAAGCAT−3 ′ ′ 5 −AAAAGCATAGAGGTACC−3 ′ ′ 5 −AAGCATAGAGGTACC−3 ′ ′ Using the overlapping regions shared between sequences, create a contig sequence of the original fragment by placing the sequence that is closest to the 5' end first and moving towards the 3' end. Answer: -5 −CTATCAAAAGCAT−3 ′ ′ -5 −ATCAAAAGC−3 ′ ′ -5 −TCAAAAGCATAGAGGTACC−3 ′ ′ -5 −AAAAGCATAGAGGTACC−3 ′ ′ -5 −AAGCATAGAGGTACC−3 ′ ′ -5 −GAGGTACCCTTC−3 ′ ′ Explanation: -Begin by looking for a region that is shared between as many fragments as possible and place that fragment with the longest 5' region first. Then, add on to the fragment from that starting point by looking for overlap in either direction. Cotyledon orbiculata, or pig's ear, is a succulent fynbos plant from South Africa whose juice is used as a treatment for epilepsy. Suppose three South African populations of C. orbiculata were collected and characterized by DNA sequencing across a sequential 8-kb interval of the nuclear genome. The data was summarized into haplotype data for each of the three populations and shows the number of individuals with each haplotype in each population and the SNPs at each collected loci. Select all of the haplotypes that are unique to a single population of C. orbiculata. Suppose the sequence characterized previously contains part of the unknown gene responsible for the medicinal properties of C. oribulata that allow the plant to treat epilepsy. Answer: -East_SA-2 -Central_SA-3 -West_SA-4 Explanation: -A haplotype is a set of DNA polymorphisms that tend to be inherited together. Haplotypes are often used in genome wide association studies to locate approximately where SNPs are found on a chromosome. Suppose that archaeologists find a tomb in Egypt containing a mummy. The tomb has been looted and any identifying hieroglyphs on the walls have worn away. The mummy does not have any visible distinguishing features, but her tissues still contain intact DNA. What could scientists do to identify who this mummy was? Answer: -Use genome analysis to compare her DNA with DNA from other identified mummies in the area Explanation: -DNA contains information about an individual's traits and is passed on from parents to offspring. Suppose a new species of thermophilic bacteria is discovered that can live in undersea volcanic vents. Researchers perform a microarray in order to determine the genetic similarities and differences between sulfur and high temperature tolerance. The results below show high gene expression in the experimental condition compared to the control as pink, low gene expression compared to the control as cyan, and no change in gene expression compared to the control as white. Each letter represents a different gene. (table here) What steps could the researchers do next in order to better understand the genes they identified? Answer: -Identify shared 5' regulatory elements in gene groups and generate reporters using the elements -Group genes in which both experimental conditions produce the same type of response -Group genes that increase in one experimental condition and decrease in the other condition Explanation: -Consider how genes can be grouped to help draw conclusions. What further testing would give you insight into how the expression of the genes is controlled? Order the steps required to analyze gene expression from a particular cell type using a DNA microarray. Answer: -extract mRNA from cells -reverse transcribe mRNA to cDNA -label cDNA with a fluorescent molecule -add cDNA to microarray and incubate -wash away unbound cDNA -visualize microarray and analyze resulting data Explanation: -In order to analyze gene expression from a particular cell type, a pool of mRNA must first be isolated from a sample. Is mRNA typically added directly to a microarray? Consider what other steps must occur first. Researchers used microarrays to examine the expression pattern of 25,000 genes from the primary tumor cells in women who had breast cancer. They determined that the gene expression patterns found in these tumor cells accurately predict the recurrence of cancer within five years after treatment. In this experiment, mRNA from cancer cells and noncancer cells was converted into cDNA and labeled with red fluorescent and green fluorescent markers, respectively. (tables here) Determine whether most of the genes represented on the microarray are overexpressed or underexpressed in cancer cells from patients who remained cancer free for five years after treatment, and select the reason why. Answer: -Underexpressed, because green indicates genes that are highly expressed in noncancer cells compared to cancer cells. Explanation: -A green dot indicates that the gene was more highly expressed in noncancer cells, whereas a red dot indicates that the gene was more highly expressed in cancer cells. After discovering a gene for the production of fur in mammals and examining the gene regulation in the genomes of a monkey, bear, zebrafish, wolf, and snake, it is evident that all of the genomes have an orthologous gene. Select the statements that show what analysis of the orthologous gene says about the function of the gene controlling the trait. Answer: -The ancestral form of the gene was potentially involved in a different biological function. -The gene function is not limited to fur production. Explanation: -All of the animals have the orthologous gene, but the zebrafish and snake do not have fur. Consider the function of the gene as it relates to expression in the zebrafish and snake. Classify each characteristic as being commonly found in bacterial genomes, in eukaryotic genomes, or in both genomes. Answer: -Bacterial genomes only: -generally have one chromosome -rarely contain introns -often contain plasmids -Eukaryotic genomes only: -have a diploid chromosome number -chromosomes are generally linear -nucleus contains chromosomes -Characteristics of both -have a DNA double-helix structure -genomes are necessary for survival -contain genes that code for proteins Explanation: -Bacteria and eukaryotes diverged from a common ancestor, so their genomes share some basic structural and functional characteristics common to all living organisms. However, bacteria are prokaryotes and therefore lack the membrane-bound organelles that eukaryotes possess. As depicted in the table, certain plant genomes are significantly larger than mammal genomes, including the human genome. Genome. Approximate Genome Size Human. 3,200,000,000 Rat. 2,750,000,000 Norway Spruce. 19,600,000,000 Whisk fern. 250,000,000,000 Why do these diploid plants have more DNA, but less organismal complexity compared with humans? Answer: -Repetitive DNA from transposons accounts for the majority of these plant genomes. How does comparative genomics aid in the search for factors that contribute to complex human conditions? Answer: -Conserved genetic sequences likely influence similar phenotypic traits across species. Explanation: -Comparative genomics involves comparing genomics features of different organisms. For example, a locus may be compared for differences between mouse and human. Which statements identify differences between proteomics and genomics? Answer: -Genomics evaluates a relatively static molecule, whereas proteomics evaluates variable molecules. -Mass spectrometry is a technique that is used in proteomics, but not in genomics. Explanation: -Proteomics is the investigation of all of the proteins in an individual, whereas genomics is the investigation of all the genes in an individual. Suppose a team of researchers sequences the genome and measures the proteome of a human skin cell and a human kidney cell. Will there be a greater difference between the genome or the proteome between these two cells? Answer: -Differences in gene expression and post-transcriptional mRNA processing lead to a greater difference in the proteome than the genome between the two cells. Explanation: -The genome is the genetic material of a cell, whereas the proteome is the complete set of proteins found within a cell. Select the ways in which mass spectrometry is used to identify proteins in a cell. Answer: -to identify the amount of each protein present in the proteome of a cell -to identify individual proteins from a database of known protein profiles Explanation: -Mass spectrometry is a technique where a molecule's precise mass and charge is measured and a profile is generated. Match each term to the most appropriate definition or example. Answer: -A trait that has a range of phenotypic variation= QUANTITATIVE TRAIT -The number of puppies in a dog's litter= MERISTIC TRAIT -The incidence of high blood pressure in a person= THRESHOLD TRAIT -A trait that is influenced by genes at multiple loci= POLYGENIC TRAIT -The amount of milk a cow produces in a day= CONTINUOUS TRAIT Explanation: -Some of the terms are subsets of other terms. Match specific terms and definitions or examples before matching the general ones. Consider how the traits are affected by genetic and environmental factors. -Kernel color in a particular strain of wheat: Kernel color is either white, light red, or dark red in this strain of wheat. Two codominant alleles, which segregate at a single locus, determine these three phenotypes. -Presence or absence of leprosy: Family pedigrees indicate that an increased susceptibility to leprosy is heritable. However, the increased susceptibility does not follow a Mendelian inheritance pattern. Repeat exposure to the bacterium that causes leprosy also increases the probability of having leprosy. -Number of branches on a species of rose bush: Several loci control the formation of 4, 6, 8, or 10 branches in this species of rose bush. This rose bush species forms more than 8 branches per bush when the rainfall is high during a growing season. -Skin color in humans: The broad range of human skin color is due to variations in the type and presence of melanin. Melanin production and accumulation is controlled by at least six genes and is also influenced by environmental factors such as nutrition and exposure to sunlight. -Number of fingers in humans: One form of polydactyly, or having extra limb digits, is caused by an autosomal dominant allele. The presence of the dominant polydactyly allele results in 6, 7, or 8 fingers per hand, instead of the normal 5 fingers. -Presence or absence of cystic fibrosis: A recessive mutation in the CFTR gene, which encodes a membrane-bound chloride channel, causes cystic fibrosis. Cystic fibrosis can be inherited from parents who do not have the disease but are both heterozygous for the recessive cystic fibrosis allele. Classify each trait to indicate whether it is a continuous trait, a meristic trait, a threshold trait, or a discontinuous trait. Answer: -Continuous trait -skin color in humans -Meristic trait -number of branches on a rose bush -Threshold trait -presence or absence of leprosy -Discontinuous trait -kernel color in wheat -number of fingers in humans -presence or absence of cystic fibrosis Explanation: -Continuous, meristic, and threshold traits are different types of quantitative traits, which are determined by both genetic and environmental factors. Discontinuous traits are determined primarily by genetic factors and show Mendelian inheritance patterns. Suppose a researcher performed an experiment to determine the number of genes affecting fruit size in tomatoes. She crossed the domestic tomato, Lycopersicon esculentum, and the wild species, Lycopersicon cheesmanii, for two generations to obtain F2 individuals. Consider the table, which reports the fruit diameter and number of offspring obtained by the researcher in the F2 generation. In the parental lines, L. cheesmanii had a fruit diameter of 7 cm7 cm and L. esculentum had a fruit diameter of 17 cm17 cm . Fruit diameter. Number of offspring