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63 Cards in this Set
- Front
- Back
What are the 3 basic elements of a plasmid vector?
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1. origin of replication;
2. selectable marker gene (often antibiotic resistant genes); 3. lacZ gene with internal MCS for selection of plasmids that have the piece of DNA of interest. (inserted DNA disrupts LacZ gene) |
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X-Gal produces ____ color if the LacZ gene is intact in the plasmid.
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blue
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How does DNA sequencing work?
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ddNTPs stop DNA polymerases. 4 mixes made for C,G,A or T reactions. Fragments are electrophoresed. The sequence is read from the bottom up of the gel and remember that the template strand will have the complementary sequence.
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ddNTPs lack an OH group on what carbon?
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3'
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Sequencing using the same oligo primer is only good for about how many base pairs?
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500 b.p.
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What is site-directed mutagenesis?
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Making mutations in the amino acid sequence to try to increase the ability of a protein.
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Is PCR DNA methylated or unmethylated?
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unmethylated.
Unmethylated DNA not cut when selecting for mutated plasmid. |
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Nettie Stevens Edmund Wilson...
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Independently described the behavior of sex chromosomes-XX determines female; XY determines male.
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Joe Hin Tjio determined that...
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the number of chromosomes in humans is 46. In 1955
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Jerome Lejune described...
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that Down syndrome is caused by trisomy 21
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The first genetic test for a disease was for what disease?
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Huntington's
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Newborn Screening
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State mandated to detect cases ASAP -
Disorders where there is treatment available Identify affected infants PRIOR to onset of irreversible findings Must be a test that can distinguish affected infants from unaffected that Can be done large scale – population screening Inexpensive, reproducible, simple, “quick” Examples: PKU, cystic fibrosis, sickle cell disease. |
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Carrier Screening
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Recessive, monogenic disorders
cystic fibrosis hemophilia Selected (at-risk) population Tay-Sachs disease Sickle cell disease |
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Hemoglobinopathies
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Appropriate screening for patients at increased risk to be carriers for hemoglobinopathies includes BOTH:
- Quantitative hemoglobin electrophoresis - CBC with red cell indices |
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When is Prenatal Testing used?
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To assess health of fetus, often when pregnancy is at increased risk of a genetic condition:
maternal age (>35) family history ethnicity abnormal multiple marker (Quad) screen Blood test during pregnancy to identify pregnancies at increased risk for certain birth defects fetal ultrasound |
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When is Diagnostic Genetic Testing used?
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Used to confirm or rule out a known or suspected genetic disorder in a symptomatic individual.
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Bifid Uvula is Associated with a number of genetic conditions including:
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22q deletion syndrome, familial cleft lip/palate, Smith-Lemli-Optiz syndrome
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Williams syndrome is caused by a Microdeletion in an Elastin gene
of Chromosome 7p. It results in: |
Cocktail party personality
Moderate mental retardation Supravulvar Aortic Stenosis Hypercalcemia Dental anomalies: Short, square teeth and may be sparsely arranged. Kidney anomalies |
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Osteogenesis Imperfecta causes:
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Multiple fractures and brittle bone
Dentinogenesis imperfecta (DI): Brittle teeth. |
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Van der Woude is...
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Most common cleft lip/ cleft palate syndrome
Autosomal dominant * Bilateral lip pits |
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Gorlin syndrome results in
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Jaw cysts and predisposition to cancers.
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Predictive Testing is Offered to...
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asymptomatic individuals with increased risk of a genetic disorder (i.e. with a family history of the disorder)
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An Example of a High Utility Predictive Genetic Test is...
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MEN2 – Multiple Endocrine Neoplasia, type 2
RET oncogene Characterized by medullary thyroid carcinomas, pheochromocytomas Childhood onset Prophylactic thyroidectomy is SOC Preventive intervention and clinically valid test |
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What is Genomics?
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The study of genomes and includes sequencing DNA and collecting genomic variations within a population as well as transcriptional control of genes
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What is Structural Genomics?
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Genome sequencing, annotation, structural organization
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What is Functional Genomics?
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Transcriptomics and mutant analysis (some include functional proteomics)
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What is Comparative Genomics?
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Comparison and studies on the evolution of genomes
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What are the limitations to DNA Microarrays?
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Expense - Genomic Arrays $100,000 - $500,000
False Positives Secondary Confirmation Upregulated expression = upregulated production???? TOOOOO much information |
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What is Proteomics?
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The scientific discipline of characterizing and analyzing the proteins, protein interactions, and protein modifications of an organism
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What is Structural proteomics?
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refers to the development and application of experimental approaches to define the primary, secondary, and tertiary structure of proteins (e.g. Edmund Deg, X-ray Crystallography, Molecular Fit Programs, etc.)
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What is Functional proteomics?
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refers to the development and application of global (proteome-wide or system-wide) experimental approaches to assess protein function by making use of the information provided by structural genomics
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What is a 1D SDS-PAGE?
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Fast, simple and reproducible tool for obtaining preliminary information on proteins
Proteins denatured and “coated” with SDS (-ve charge) then pushed through a polyacrylamide gel via electric current MW proportional to 1/Speed of mobility |
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Mass Spectrometry
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Two Components:
1. Ionization and Separation: Matrix assisted laser desorption ionization (MALDI) Electrospray ionization (ESI) 2. Mass Spectrometry: MALDI – combined with Time of Flight (ToF) ESI – combined with quadrupole MS Derivation of Peptide Sequence |
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MALDI-ToF MS
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Matrix – α-cyano-4-hydroxycinnamic acid or DHB
Laser desorption – N2 lasers 337nm Ionization via photons (a little rough so small molecules to peptides) Time of Flight: Ions collected in a vacuum between two plates charged at 20,000 V Cut power to one plate allows ions to travel down tube to a detector and the time of flight is recorded ToF is proportional to m/z or MW |
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What are the benefits of Mass Spectrometry?
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Automation
Low sample volume (nL) Rapid (minutes) Sensitive (nano-femtomoles) Quantitation possible (area under the peak) Multisamples (384) |
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What is Metabolomics?
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The "systematic study of the unique chemical fingerprints that specific cellular processes leave behind" - specifically, the study of their small-molecule metabolite profiles
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What is a Metabolome?
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the collection of all metabolites in a biological organism, which are the end products of its gene expression
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Metabolic profiling can give an instantaneous _______ of the physiology of that cell.
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'snapshot'
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What percentage of the US population is diagnosed with cancer during their lifetime?
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25%
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How many new cancer patients are diagnosed each year?
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1.3 million
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~75% of these patients receive systemic chemotherapy, however it will only provide a cure or long term remission in how many of these cases?
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10%
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What is the overall 5 year survival rate for cancer?
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65%
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As a cause of mortality, cancer is second only to...
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cardiovascular disease
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What are the criteria that distinguish transformed cells from normal cells in culture?
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1. the requirement for serum in the cell culture medium to stimulate growth. Transformed cells have a reduced requirement for serum, approximately 10% that required for normal cells to grow.
2. the ability to grow without attachment to a supporting matrix (anchorage dependence). Normal cells (such as fibroblasts, smooth muscle cells) require adherence to a substratum (in this case, the bottom of the plastic dish) and will not grow if suspended in a soft agar mixture (the consistency) of loose jello. Transformed cells, however, have lost this anchorage dependence for growth |
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The test used to screen a chemical for potential carcinogenicity is called...
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Ames assay
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How does the Ames assay work?
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(1) Basis for test: Salmonella typhimurium with mutations (His-) unable to code for one enzyme in histidine synthetic pathway, thus needs histidine in medium(2) Procedure: test chemical for potential to cause mutation at His- site changing it to His+ allowing bacteria to grow without histidine in medium
(3) Problem: bacteria not contain spectrum of monooxygenases found in higher animals, thus compound requiring activation to become mutagenic not occur in bacteria(a) Solution: incubate agents to be tested with postmitochondrial supernatant of rat liver (S-9 fraction) which contains various enzymes to activate potential mutagens & carcinogens |
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The genes involved in the development of cancer are classified as...
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oncogenes or tumor suppressor genes
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mutated derivatives of normal genes whose function is to promote proliferation or cell survival
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Oncogenes
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proto-oncogenes can code for:
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A. Growth factors
B. Growth factor receptors C. Signal transduction proteins D. Intracellular kinases E. Transcription factors |
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Mutant p53 is present in what percent of cancers?
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75%
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P53 is located on the small arm of ________ and is present in all normal tissues
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Chromosome 17
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the most common genetic change in human cancer is what?
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Mutation of p53
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When a mutant allele inactivates the normal one, it is called a...
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dominant negative gene
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p53 is an important tumor suppressor and it works what 3 ways?
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Preventing division of cells:
Arrests cells in G1 stage to allow time for DNA repair Triggers apoptosis: If irreparable, triggers apoptosis Production of regulatory molecules: helps slice specific RNA into small regulatory molecules called microRNAs |
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Most mutations occur in the ________ domain of p53
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DNA-binding
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Does p53 play a role in primary RNA transcript production?
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No.
p53 mutations disrupt the machinery that chops RNA into microRNA |
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p53’s DNA-binding domain interacts with what other two proteins?
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Drosha and p68 are proteins in an assemblage responsible for snipping primary transcripts into hairpin-shaped molecules
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True/False?
Mutant p53 is worse than no p53 |
True
Normal p53 serves to brings Drosha and p68 together Altered p53 keeps the two apart and interrupts their normal interaction |
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True/False?
A cell population must accumulate multiple mutations for transformation to malignant cells. |
True
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Apoptosis is initiated by either
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death receptor activation
or intracellular signals leading to release of the mitochondrial protein, cytochrome c. |
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Around ____ of the deaths in the United States each year are caused by cancer
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20%
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tumor-generating cells share key traits with _____ cells, including an unlimited life span and the ability to generate a diverse range of other cell types
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stem
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Acute myeloid leukemia (1994)
Acute lymphoblastic leukemia (1997) Chronic myelogenous leukemia (1999) Breast (2003) Multiple myeloma (2003) Brain (2004) Prostate (2005) |
malignant stem cells have been identified to date
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