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48 Cards in this Set
- Front
- Back
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Recombinant DNA Techniques:
Strategies to obtain fragments of DNA and copies of genes- |
-restriction fragments
-DNA produced by reverse transcriptase -Chemical synthesis of DNA |
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Recombinant DNA Techniques:
Techniques for identifying DNA seqences- |
-probes
-Gel electrophoresis -detection of specific DNA sequences -DNA sequencing |
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Recombinant DNA techniques:
Techniques for amplifying DNA sequences- |
-cloning of DNA
-libraries (dCNA & genomic) -Polymerase Chain reaction (PCR) |
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What is recombinant DNA?
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DNA combined from 2 different sources
Assembly of recombinant DNA molecules requires molecular cloning |
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Advantages of using bacteria for recombinant DNA
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-easy to grow
-reproduce quickly & asexually -daughter cells contain exact same DNA as parent -easy to isolate DNA -easy to get DNA back into bacteria (transformation) |
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Sources of DNA
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-genomic DNA
-cDNA (amplified using PCR) -genomic fragments (amplified using PCR) -synthetic oligonucleotides |
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What do vectors contain?
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MCS and gene of antibiotic resistance
-plasmid, virus, or artificial chromosome -can be used for cloning |
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What are restriction endonucleases and what do they do?
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-bacterial enzymes that cut foreign DNA
-named after organism derived from -recognize & hydrolyze specific sequences - |
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What sequence does EcoRI recognize and cleave?
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-Palindromic site
-GAATTC, cleaves btwn G & A on both strands of DNA -produces staggered end fragments |
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What does reverse transcriptase do?
What do DNA polymerases do? |
-Reverse transcriptase converts RNA to cDNA
-DNA polymerases fill cohesive ends & synthesize second strand of cDNA |
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DNA polymorphism
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-variations among individuals of a species in DNA sequences of genome. could be point mutations, deletions, & insertions
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Detection of polymorphism
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-restriction fragment length polymorphism (RFLP)
-testing of mutations by PCR -detection of polymorphism caused by repetitive DNA -DNA chip microarrays |
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Vaccines
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-recombinant antigenic proteins expressed on surface of pathogens are used to make vaccines (hepatitis B)
-DNA vaccines are used to enter host cell, DNA produces antigen from organism against which antibody production is desired -expressed protein generates immune response to antigen & host protection |
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Production of therapeutic proteins
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Insulin:
-2 protein chains comprising isulin produced in E. coli seperately -chains purified, mixed, & refolded to produce active isulin Human growth hormone: -also produced in E. coli, used to treat children w/ growth deficiencies |
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Genetic counselling (process)
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-evaluating family history & medical records
-ordering genetic tests -evaluating results of investigation -helping parents understand & reach decisions for next step |
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Gene therapy
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-use of DNA as a pharmeceutical agent to treat disease
--replace a mutated gene w/ healthy one --inactivate mutated gene that is functioning improperly --introduce new gene into body to fight disease |
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Transgenic animals
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-animals that carry foreign genes that have been deliberately inserted into it's genome
-2 methods (mice): 1. inject gene into pronucleus of fertilized mouse egg 2. transforming embryonic stem cell growing in cell culture w/ desired DNA |
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Proteomics
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comprehensive analysis & characterization of all the proteins & protein isoforms encoded by the human genome
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What do the following cloning enzymes do?
DNA ligase Phosphatase Kinase |
DNA ligase- links DNA fragments together
Phosphatase- removes phosphate from 5' end of DNA Kinase- adds phosphate to 5' end of DNA or RNA |
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Process of reverse transcriptase (5 steps)
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1. total RNA extracted
2. Poly-T oligonucleotide used as primer for DNA synth. by reverse transcriptase 3. RNA in RNA/cDNA hybrid cleaved by RNase H 4. second strand DNA synthesized by DNA polymerase using RNA fragments as primer 5. double-stranded cDNA used for cloning, amplification, or sequencing |
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Define probes
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-single stranded DNA or RNA molecules used to detect complementary DNA or RNA sequence
-they carry radioactive or chemical marker to facilitate detection |
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define hybridization
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-very specific & sensitive
-able to detect on molecule per cell -can be used to determine # of copies of DNA sequence in sample |
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Process of hybridization
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1. heat or alkali added to dsDNA
2. ssDNA results & probe w/ label added 3. probe hybridizes only w/ complementary sequence on DNA, allows sequence identification |
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What is FISH and what is it used for?
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-floursecent in situ hybridization (FISH)
used to: -identify numerical & structural chromosome abnormalities -track origin -characterize marker cells -identify regions of deletion or amplification |
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What is gel electrophoresis & what is it used for?
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-method for seperation & analysis of macromolecules
-acrylamide gel used to seperate DNA fragments (100-25,000 nucleotides long) that differ in length by only 1 nucleotide -denaturing used for ss DNA but not ds DNA |
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How do DNA fragments seperate in gel electrophoresis?
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-nucleic acid is neg so it migrates from neg cathode to pos anode
-shorter fragments move faster |
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after fragment seperation using gel electrophoresis, how can specific sequences be detected (RNA and DNA)?
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RNA- using northern blot
DNA-using southern blot |
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Detection process for bacterial colonies
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1. transfer colonies to paper
2. denature DNA w/ alkali 3. Add readioactive probe, incubate & wash out non-hybridized probe 4. autoradiography 5. identify positive colonies |
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Detection process for DNA & RNA
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1. separate DNA or RNA on agarose gel
2. transfer seperated nucleic acids on nitrocellular paper 3. remove nitrocellulose paper from gell 4. hybridize seperated nucleic acids w/ radiolabelled probe 5. visualize probe hybridized to complementary DNA by autoradiography |
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methods of DNA sequencing
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Maxam-Gilbert: uses chemical degradation of DNA at specific bases
Sanger: sequencing uses termination of DNA synthesis at specific nucleotides Next generation sequencing (NGS) platforms: ion torrent, 454, allumina |
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Diff btwn methods
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Maxam-Gilbert- more labor intense than sanger, uses radiolabels & hydrolysis, followed by gel electrophoresis & autoradiography
Sanger- requires ss DNA, primer, & labelled ddNTP, uses autoradiography if radiolabelled ddNTP used NGS- immobilize DNA onto beads, detects light or H ions |
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define genomics
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used of NGS to ***** personal risk of having or developing certain diseases
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define metabolomics
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analysis of metabolites in urine to predict drug metabolism
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define pharmacogenomics
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field of study examines impact of genetic variation & drug responses by biomarker (medicine)
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Cloning of DNA
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1. DNA inserted into plasmid containing antibiotic resistance gene
2. Transform bacterial cells 3. Select colony containing plasmid by growing on the medium w/ antibiotic 4. grow bacteria from selected clone 5. isolate plasmid DNA from bacterial culture |
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What is PCR (polymerase chain reaction)?
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enzymatic synthesis of identical DNA fragment repeated many times, products of previous cycle used at templates in the following cycle
1. heat to separate strands 2. hybridization of primers 3. DNA synthesis from primers (in-vitro) -allows for rapid production of very large amounts of specific segment DNA |
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How does DNA fingerprinting work?
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-human DNA contain sequences that are repeated many times at certain loci in genome
-regions contain VNTR -restriction enzymes recognize sequences that flank VNTR region and produce restriction fragments -special probes identify fragments, fragment size is unique to each individual |
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Three genetic parent embryo
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-central nucleus w/ DNA from mother & father
-mitochondrial DNA from another "mother" |
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what are biomarkers?
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biological molecules found in blood, other body fluids, or tissues that are a sign of a normal or abnormal process, or of a condition or disease
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SDS-page seperates proteins based on what?
How are these proteins visualized? |
molecular weight
visualized w/ western blot, using protein-specific antibodies |
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How does mass spectrometry work?
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1. inject sample
2. heater vaporizes sample 3. electron beam ionizes sample 4. particles accelerated into magnetic field 5. magnetic field separates particles based on mass/charge ratio |
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How does isoelectric focusing work?
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uses pH to seperate proteins based on their isoelectric point
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What is immunoblotting/western blot?
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technique to seperate proteins
1, seperated by isoelectric focusing (charge) 2, separated by SDS-Page (MW) 3. transferred to nitocellulose 4. nitrocellulose incubated w/ antibodies that recognize proteins phosphorylated on threonine residue 5. position of protein recognized by antibodies revealed by enzyme-linked secondary antibody |
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How does microbiota benefit it's human host?
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- contributes to digestion
- metabolism of xenobiotics - development of immune system - colonization resistance *beneficial only during homeostasis |
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***Technique & application:
Different blots- western, northern, southern |
Western & northern for RNA
Southern DNA |
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***What are they detected for (diff molecules)? All similar technique
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***Technique for identification/isolation of mutation/protein
Choose best way |
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***RtPCR, PCR, sequencing, microarray, fish, all used for diff purposes, best quickest most efficient way
Slides about metabolizers/drugs are not going to be tested What drug is better not tested |
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