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BMSC 210 Midterm 2 | 100% Correct
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What is genetic engineering? - ✔✔using in vitro techniques to alter genetic material in the lab What is recombinant DNA technology? - ✔✔the artificial recombination of DNA from two organisms What are some basic techniques of genetic engineering? - ✔✔ DNA amplification electrophoesis nucleic acid hybridization molecular cloning expressing foreign genes targeted mutagenesis What is heterologous expression? - ✔✔expressing a gene in a different host What is the polymerase chain reaction? - ✔✔rapid amplification in the number of copies of specific DNA sequences for further analysis What is a thermocycler? - ✔✔an automated PCR machine What is quantitative PCR? - ✔✔quantifies initial amount of DNA What are some applications of PCR? - ✔✔-obtain specific fragments of DNA for cloning
- comparative (phylogenic) studies to amplify genes and DNA sequences
- amplify VERY small amounts of DNA (forensic)
What are the steps in PCR amplification? - ✔✔1. DNA is denatured by heating
- Synthetic piece of DNA flanking sequence of interest is added
- Add DNA polymerase (Taq or Pfu polymerase)
- heat and cool - repeat Why is use of thermostable DNA polymerase critical? - ✔✔because of high temperatures What do PCR require? - ✔✔DNA polymerase and artificial oligonucleotide primers made of DNA What is reverse transcription PCR? - ✔✔can make DNA from an mRNA template What enzyme is used in reverse transcription PCR? - ✔✔reverse transcriptase
- converts RNA into complementary DNA What is gel electrophoresis? - ✔✔the separation of charges molecules using an electric field What are agarose gels stained with? - ✔✔ethidium bromide DNA cut with different restriction enzymes will have the same or different banding patterns on an agarose gel? - ✔✔different What is nucleic acid hybridization? - ✔✔Single strands of DNA, or of DNA and RNA, from related organisms will hydrogen bond to form a double stranded molecule What are nucleic acid probes? - ✔✔ss DNA fragment complementary to the gene of sequence of interest How are nucleic acid probes created? - ✔✔cloning, synthesis, denaturing a fragment of DNA
What are palindromes? - ✔✔recognize inverted repeat sequences What is a palindromic sequence? - ✔✔a nucleic acid sequence (DNA or RNA) that is the same whether read 5' to 3' on one strand or 5' to 3' on the complementary strand What is EcoRI? - ✔✔a restriction endonuclease enzyme that is isolated from strains of ecoli What does the nucleic acid sequence enzyme cut in ecori? - ✔✔GAATTC What does ligation do for ecori? - ✔✔ligation by DNA ligase can rejoin the two sugar-phosphate backbones of DNA through covalent binding Why DNA modification? - ✔✔each restriction enzyme is partnered with a corresponding modification enzyme that shares the SAME recognition sequence Restriction endonucleases - ✔✔enzymes used for cloning DNA ligase - ✔✔catalyzes the joining of two strands of DNA b/w the 5' P and 3' OH of adjacent nucleotides with either cohesive or blunt ends Reverse transcriptase - ✔✔converts RNA into DNA DNA polymerase - ✔✔mostly used for 5'3' polymerizing activity (also 3'5' and 5'3' exonuclease) Plasmids - ✔✔natural vectors and are useful for cloning vectors Why is pUC19 a good cloning vector? - ✔✔bc it has all essential elements of a cloning vector is a modified ColE1 plasmid containing: ampicillin resistance & lacZ genes and polylinker within lacZ
Insertional inactivation - ✔✔vectors with lacZ genes, lacZ is inactivated by insertion of foreign DNA and B-galactosidase NOT produced Blue/white screening (bacteria) - ✔✔bacteria with cloning vector which may or may not contain "insert" DNA Blue colonies of bacteria - ✔✔DO NOT have vector with foreign DNA inserted White colonies of bacteria - ✔✔HAVE foreign DNA inserted Initial selection - ✔✔-antibiotic resistance
- often sufficient enough for cloning of PCR- generated DNA sequencing Antibodies to detect correct clones - ✔✔blood serum proteins produced by animals after injection with a specific protein antigen, can be fluorescently labelled Yeast artificial chromosomes (YACs) - ✔✔-used for cloning into yeast
- linear vectors that replicate like normal chromosomes but have sites for insertion of large DNA fragments Common host strains for cloning - ✔✔-E coli
- Bacillus subtilis
- Saccharomyces cerevisiae Ideal hosts should be: - ✔✔-capable of rapid growth
- non-pathogenic
- capable of incorporating DNA
- genetically stable
- have correct enzymes
Green Fluorescent Protein (GFP) - ✔✔a protein that exhibits bright green fluorescence when exposed to blue light Genomes - ✔✔entire complement of genetic infor
- includes genes, regulatory sequences, and noncoding DNA Genomics - ✔✔Discipline of mapping, sequencing, analyzing, and comparing genomes RNA virus MS2 - ✔✔first genome sequenced in 1976 3,569 bp Haemophilus influenzae - ✔✔-first cellular genome sequenced in 1995
- 1,830,137 bp What can genomes tell us? - ✔✔- heat-stable enzymes to virulence factors
- solves medical mysteries Sequencing of genomes - ✔✔determining the precise order of nucleotides in a DNA or RNA molecule Generation in genomes - ✔✔refers to successive major changes in sequencing technology that confer Sanger method - ✔✔first generation sequencing First generation DNA sequencing - ✔✔-Fred Sanger
- chain termination method Second generation DNA sequencing - ✔✔-Generates data 100x faster than Sanger method
- massively parallel methods
454 sequencing system - ✔✔-DNA is broken into small segments
- DNA is amplified using polymerase chain reaction (PCR)
- Light is released each time a base is added to DNA strand
- Instrument actually measures release of light
- Can handle only short stretches of DNA Shotgun DNA sequencing - ✔✔entire genome is cloned, and resultant clones are sequenced (redundant)
- virtually all genomic sequence is this Genome assembly - ✔✔consists of connecting the DNA fragments in the correct order and eliminating overlaps (usually done by computer) Closed genome - ✔✔Entire gene sequence obtained Open genome - ✔✔some small gaps Annotation of genomes - ✔✔converting raw sequence data into a list of genes present in the genome
- "bottleneck" in genomics Bioinformatics - ✔✔science that powerful computational tools to DNA and protein sequences for the purpose of analyzing, storing, and accessing the sequences for comparative purposes Hypothetical protein - ✔✔uncharacterized ORFs; proteins that likely exist but whose function is currently unknown; likely encode nonessential genes; in E. coli, many predicted to encode regulatory or redundant proteins Noncoding RNA - ✔✔RNAs that do not encode proteins and is not translated, but have important regulatory functions
- lack start codons
Mobilome - ✔✔the mobile genetic elements in a genome, which shuffles genes b/w species Transposons - ✔✔mobile genetic elements that move b/w chromosome, plasmids, and viruses via transposase Insertion sequences - ✔✔simplest transposable elements provided by diversity Cor genome - ✔✔shared by all strains of the species Pan genome - ✔✔includes all the optional extras present in some but not all strains of the species Chromosomal islands - ✔✔clusters of genes for specialized functions not essential for survival Metagenomics - ✔✔entire complement of RNA (transcriptome), proteins (translatome), or metabolites (metabolome) produced under certain conditions Mycobiome - ✔✔the total complement of fungi in a natural environment Metagenome - ✔✔The total gene content of the organisms present in an environment "biome studies" Transcriptome - ✔✔The entire complement of RNA produced under a given set of conditions Microarrays - ✔✔Small solid-state supports to which genes or portions of genes are fixed and arrayed spatially in a known pattern What info can be derived from microarrays? - ✔✔•Global gene expression •Expression of specific groups of genes under different conditions •Expression of genes with unknown function; can yield clues to possible roles •Comparison of gene content in closely related organisms
•Identification of specific organisms RNA-Seq - ✔✔Replacing microarrays for the analysis of gene expression Proteomics - ✔✔Genome-wide study of the structure, function, and regulation of an organism's proteins Proteome - ✔✔all the proteins encoded by the genome or only present at a given times Proteins with >50% sequence similarity - ✔✔typically have similar functions Proteins with >70% sequence similarity - ✔✔almost certainly have similar functions Protein domains - ✔✔-Distinct structural modules within proteins
- Have characteristic functions that can reveal much about a protein's role, even in the absence of complete sequence homology Interactomes - ✔✔complete set of information of protein-protein interactions (MALDI & TOF) MALDI - ✔✔matrix assisted laser desorption ionization TOF - ✔✔time of flight Plant biochem - ✔✔plants produce several thousand metabolites secondary metabolites - ✔✔scents, flavours, alkaloids, pigments Systems of biology - ✔✔the integration of data from genomics and other "omics" areas to build an overall picture of a biological system
nucleocapsid - ✔✔complete complex of nucleic acid and protein packaged in the virion enveloped virus - ✔✔virus that contains additional layers around the nucleocapsid helical symmetry - ✔✔-Rod-shaped viruses
- Length of virus determined by length of nucleic acid
- Width of virus determined by size and packaging of protein subunits icosahedral symmetry - ✔✔spherical viruses; most efficient arrangement of subunits in a closed shell enveloped viruses - ✔✔have an outer lipid bilayer membrane surrounding nucleopcapsid and embedded with proteins Influenza virus - ✔✔the envelope contains rigid "spikes" of haemagglutinin and neuraminidase which form a characteristic halo of projections around negatively stained virus particles ebola virion - ✔✔-helical
- spikes of envelope
- lipid envelope
- mem-associated proteins
- nucleocapsid proteins complex viruses - ✔✔virions composed of several parts, each with separate shapes and symmetries lysozyme like enzyme - ✔✔-makes hole in cell wall to allow nucleic acid entry
- lyses bacterial cell to release new virions Nucleic acid polymerases enzymes - ✔✔-reverse transcriptase
- RNA virus - RNA dependent RNA polymerase (RNA replicase)
Neuraminidases (influenza virus) enzymes - ✔✔-enzyme that cleave glycosidic bonds (destroys glyco- lipids/pro)
- allows liberation of viruses from cell Growth of viruses - ✔✔-flasks used to culture human or animal viruses
- plates containing T4 grown on ecoli lawn, clear plaques indicate lysis of cells What viruses are the easiest to grow? - ✔✔bacterial viruses Titer - ✔✔number of infectious units per volume of fluid plaque assay - ✔✔analogous to the bacterial colony; one way to measure virus infectivity plaques - ✔✔are clear zones that develop on lawns of host cells each plaque results from infection by a single virus particle Efficiency of plating - ✔✔- used in quantitative virology
- The number of plaque-forming units is almost always lower than direct counts by electron microscopy What are the phases of viral replication? - ✔✔attachment entry synthesis assembly release latent period - ✔✔eclipse + maturation
Packing of T4 genome - ✔✔-precursor of bacteriophage head assembled
- packaging motor assembled
- doublestranded DNA pumped to head
- DNA, T4 tail, tail fibers added Proheads - ✔✔bacteriophage head precursors Replication cycle of bacteriophage T4 - ✔✔~25 minutes and bust size of ~100 virions virulent - ✔✔Viruses that always lyse and kill host after infection temperate - ✔✔viruses replicate their genomes in tandem with host genome and without killing host, establishing long-term, stable relationship lysogen - ✔✔-host cell that harbors temperate virus
- a bacterium containing a prophage temperate viruses - ✔✔can undergo a stable genetic relationship within the host, but can also kill cells through lytic cycle lysogeny - ✔✔state where most virus genes are not expressed and virus genome (prophage) is replicated in synchrony with host chromosome prophage - ✔✔viral DNA repressor protein - ✔✔a regulatory protein that binds to an operator and blocks transcription of the genes of an operon Two differences b/w animal and human viruses - ✔✔1. entire virion enters the animal cell
- eukaryotic cells contain a nucleus, the site of replication for many animal viruses virulent infection - ✔✔lysis of host cell, most common latent infection - ✔✔Viral DNA exists in host genome and virions are not produced; host cell is unharmed unless/until virulence is triggered. persistent infection - ✔✔Release of virions from host cell by budding does not result in cell lysis. Infected cell remains alive and continues to produce virus transformation - ✔✔conversion of normal cell into tumor cell cell fusion - ✔✔two or more cells become one cell with many nuclei retroviruses - ✔✔RNA viruses that replicate through a DNA intermediate gag - ✔✔encode structural proteins pol - ✔✔encode reverse transcriptase and integrase env - ✔✔encode envelope proteins
