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131 Cards in this Set
- Front
- Back
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thcut DNA at specific recognition sequences, producing fragments joined with DNA vectors to form recombinant DNA molecules
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restriction enymes
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replicate autonomously in host cells & facilitate the manipulation of newly created recominant DNA molecules
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vectors
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vectors are constructed from
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often bacterial plasmids and phages
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host cells commonly used for replication
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bacteria, yeast, mammalian cells
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method for amplifying a specific DNA sequence present in a collection of DNA sequences (e.g. genomic DNA)
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polymerase chain reaction
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the PCR method allows DNA to be cloned without
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host cells
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DNA sequence analysis methods
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restriction mapping, DNA sequencing, Southern blotting
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uses hybridization to identify genes and flanking regulatory regions within cloned sequences
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Southern blotting
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In 1971, Danna and Nathans described the
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isolation of an enzyme from a bacterial strain & use of the enzyme to cleave viral DNA at specific nucleotides sequences
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recombinant DNA
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formed by joining two heterologous molecules - usually produced by in vitro ligation of DNA from two different organisms
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restriction enzymes (endonucleases)
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generate specific DNA fragments in a sequence - cleaves or nicks DNA at those sites, derived from a variety of microorganism
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human genome contains
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more than 3 billion nucleotides and 25 000 to 30 000 genes
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restriction fragments' size depends on
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how often a given restriction enzyme cuts the DNA -
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if all 4 nucleotides are present in equal proportions enzymes with a four base recognition sequence will cut how many times?
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on avg. every 256 base pairs (4^n - 4^4=256) - this is many small fragments
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NotI hase an ___ base recognition sequence while AluI has a ____ sequence.
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NotI
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why do actual fragment sizes produced by cuting DNA with a restriction enzyme vary?
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number and location of recognition sequences are not always distributed randomly in DNA
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palindromes in DNA typical of
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recognition sequences can be read from either strand when read in the 5' to 3' direction
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recombinant vectors
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vectors carrying an inserted fragment
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one of the first restriction enzymes to be identified
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from E. coli strain R - EcoRO w/six-nucleotide recognition sequence
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DNA fragments produced by EcoRI digestion have
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overhanging single-stranded tails "sticky ends" that can form hydrogen bonds with complementary single-stranded tails - once bonded can be covalently linked to form recombinant DNA molecules
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plasmid vectors were
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the first vectors developed; from naturally occuring extrachromosmal double-stranded DNA molecules that replicate autonomously w'in bacteria
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plasmid vectors are modified to
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increase their copy number so can get several hundred copies & engineered to contain convenient restriction enzyme recognition sequences & marker genes so can identify them in cells
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pUC18 is? advantages?
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a plasmid vector; small so it can carry large DNA inserts, has an origin of replication & produces up to 500 copies of fragments per cell, has polylinker in a lacZ gene
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polylinker
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engineered region of vector containing large number of restriction enzymes
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how does the polylinker of pUC18 aid in identification
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in a lacZ gene - disrupts the lacZ gene resulting in white colonies (normal expression give blue colonies) when grown on Xgal
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lambda phage
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genetically modified strains are used as vectors to carry pieces of DNA larger than 10kb - ~ 20kb. Genome has een completely sequenced and mapped - middle 3rd can be replaced w'out affecting ability to infect cells & form plaques
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how do you clone DNA using a lambda vector?
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purify and cut phage DNA using restriction enzyme (e.g. EcoRI) - cut into 3 chromosomal fragments: L&R arms and disposable middle
arms isolated & mixed with foreign DNA cut with same enzyme DNA ligase produces recombinant vectors vectors packaged into phage protein heads in vitro & added to bacterial hosts after replication creates plaques by rupturing host |
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what is a plaque
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clear area on otherwise opaque bacterial lawn caused by growth & reproduction of phages from which cloned DNA can be recovered
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cosmid vector
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hybrid vectors from combining parts of lambda chromosome with parts of plasmids - contain the cos sequence of phage lambda, pplasmid-derived antibiotic resistance gene
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what is the cos sequence of phage lambda
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necessary for packaging DNA into phage protein coats and for replication
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cosmids replicate as
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plasmids
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cosmids can carry DNA inserts much larger than lambda vectors (50 kb vs. 10-15) because
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most of the lambda genome has been removed
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shuttle vectors
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e.g. animal viruses like SV40 - hybrid vectors - contain genetic markes that are selectable in boh types of host cells and used to shuttle DNA inserts between E. coli & another type of host cell (e.g. yeast)
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bacterial artificial chromosome (BAC)
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a vector with a large cloning capacity based on the fertility plasmid (F factor) of bacteria used to act as vectors for eukaryotic DNA & can carry 300 kb inserts
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F factors are capable of
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capable of independently replicating plasmids that transfer genetic information during bacterial conjugation - carry fragments p to 1 Mb in length
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BAC vectors contain
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F factors genes for replication & copy number
at least 1 antibiotic resistance marker polylinker containing clustered recognition sequences polylink flanked by promoter sequences used to generate RNA molecules for the expression of the cloned gene for use as probes in chromosome walking & sequencing the insert |
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expression vectors
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engineered to produce many copies of a selected protein in a host cell - avail. for both pro & eukaryotes e.g. pET for use in E coli host
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use of pET expression vector
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gene to be copied clined into a recognition sequence in the polylinker next to T7 viral promotor and lac operon
host cell genome modified to carry T7 vral polymerase, lac promotor and he lac operator expression induce by adding lactose analog IPTG to medium, displacing repressor from lac operator, activating T7 polymerase gyrase gene on the bacterial chromosome AND the gene in polylinker T7 polymerase binds to the T7 promotor & transcribes the gene in the polylinker |
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IPTG
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analog of lactose placed in medium to displace repressor from lac operator leading to transcriptioon of inserted protein-coding fragments in pET
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T7 polymerase
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activated by IPTG activity on the lac operon - binds to T7 promotor & transcribes gene in polylinker of expression vector IPTG
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4 steps required to create recombinant DNA molecules & transfer to E coli host
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1. isolation of DNA to be copied, treatment w/ enzyme to make fragments
2. fragments ligated to plasmids cut with same enzyme - recombinant vector 3. vector transferre to E. coli when replicates 4. bacteria lated on nutrient medium, form colonies, screened for presense of recombinant plasmids |
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K12
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laboratory strain of E. Coli that is genetically well characterized & can host wide range of vectors - cells in colonies derived from single ancestral cell so all cells in coliny & teir plasmids are genetically identical
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phages containing foreign DNA are used to infect host E coli - when plated, each resulting plaque represents _____ .
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a cloned descendant of a single ancestral bacteriophage
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Saccharomyces cerevisae
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yeast widely used as host for cloning & expression of eukaryotic genes
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5 reasons to use yeast as a eukaryotic host
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1. can be grown much like bacteria
2. genetics widely studied - large catalog of mutations good gene map 3. entire genome has been sequenced & most genes ientified 4. can posttranslationally modify proein to it folds into fxn'l form to study fxn - bacteria can't 5.safe organism for producing protin for vaccines & therapeutic agents |
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examples of recombinant proteins synthesized in yeast
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Hep B virus surface proein
malaria parasite protein epidermal growth factor platelet-derived growth factor alpha1-antitrypsin clotting factor XIIIA |
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yeast artificial chromosome (YAC)
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a yeast cloning vector - has telomeres at each end, an origin of replication & a centromere joined to slectable marker genes TRP1 & URA3 + a cluster of respriction enzyme recognition sequences
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yeast chromosomes can carry ______ & so are useful to genome projects
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100-1000 kb in YACs
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transformation
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DNA transfer when vector is a plasmid
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transfection
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DNA transfer using a viral vector
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agrobacterium tumifaciens
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soil bacterium - infects plant cells and produces tumors (plant galls) in many plants - source of Ti plasmid - used to transfer recombinant
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plant cells carrying a recombinant Ti plasmid can be grown in tissue culture to form
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a cell mass called a callus that can be induced to form roots & shoots & eventually a mature plant carrying a foreign gene
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T-DNA
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section of the Ti plasmid (tumor-inducing) from A. Tumifaciens that control tumor formation & compounds required for growth of infecting bacteria - foreign genes can be inserted here & placed in plants by infection with A. tumifaciens
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transgenic organisms
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contain foreign gene(s)
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methods for transferring DNA into mammalian cells
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endoytosis, YACs, infection be retrovirus vectors - usually DNA then integrated into host genome
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advantage of YACs as vectors for mammalian cells
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increases efficiency of gene transfer into mice for research - transfer YAC into nucleus of fertilized egg or stem cell - makes transgenic mice
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dsDNA
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double-stranded DNA
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how do retroviral vectors function?
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single-stranded RNA transcribed by reverse transcriptase into a dsDNA molecule that integrates into host genome & is passed to daughter cells - virus can be engineered to remove viral genes so can accept forieng DNA including human
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one application of retrovirus-based vectors
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gene therapy
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polymerase chain reaction
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developed by Karl Mullis - rapid method of DNA cloning that eliminates need for host cells
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how PCR copies specific DNA sequences
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use sequence of desired gene to synthesize two oligonucleotide primers (one for 5', one for 3' end) - primers bind to complementary nucleotides flanking tge sequence to be cloned - heat-stable polymerase added after hybridization to synthesize second strand of DNA; repeat
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3 steps of PCR
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1. denaturation
2.primer annealing 3.extension by polymerase |
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primer annealing
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temperature lowered to between 50-70 deg. C (annealing temperature)so primers bind to single strands flanking DNA to be copied
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denaturation of DNA -
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heat 90-95 C - DNA dissociates into single strands in about 5 min
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Taq polymerase
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heat-stable form of DNA polymerase added to primed single-stranded DNA - DNA synthesis carried out 70-75 C - extends the primers
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extension (in PCR)
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Taq polymerase extends primers by adding nucleotides in the 5' to 3' direction, making double-stranded copy of target DNA
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advantages of PCR over cell-based cloning
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rapid - takes a few hours, not days
design of primers can be done with computer commercial synthesis of oligonucleotides is fast & cheap sensitive & amplifies from tiny DNa smaples - can use samples that are partially degraded or embedded in a medium e.g. amber |
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uses for PCR
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genetic testing, forensics, moleclar paleontology
screening for mutations in genome, identifying markers, can synthesize allele-specific probes for genetic testing |
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limitations of PCR
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info about nucleotide sequence of target must be known
minor contamination of sample with DNA from other sources causes problems e.g. shed skin cells from cop at crime scene |
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genomic library
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contains at least one copy of all the sequences in a genome, constructed using host cell cloning sine PCR fragments are small
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how big does a genomic library have to be to have 95-99% chance og containing all sequences in a genome?
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N=ln(1-P) / ln(1-f)
where N is number of required clones P probability of recovering a given sequence f is fraction of genome in each clone |
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flow cytometry
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technique used to prepare cloned libraries from individual human chromosomes
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how does flow cytometry work?
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chromosomes isolated by staining metaphse chromosomes with 2 fluorescent dyes for AT and GC pairs - flow past a laser beam that causes them to flouresce & photometer sorts & fractios by dye binding & light scattering
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pulsed-firled gel electrophoresis
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version of gel electrophoresis used to isolate chromosomes for chromosome-specific libraries
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unexpected discovery from sequencing chromosome III of yeast
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~50% of genes on this chromosome were unknown - showed mutagenesis and traditionally gene mapping very inefficient
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cDNA library
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contains DNA copies make from mRNA molecules present in a cell population at a given time, representing genes transcriptionally active at the time - isolates poly-A 3' tail bearing mRNA on
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why is cDNA so named
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complementary to the mRNA active in cell population at the time
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how are clones in a cDNA library different from genomic library
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introns are removed, mRNa doesn't include sequences near gene that regulate it
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steps in producing cDNA
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mixing mRNAs with poly-A tails with oligo-dT primers to form a partially double-stranded product that is extended by reverse transcriptase to form a complementary DNA copy - mRNA-DNA double-stranded hybrid acted on by RNAase H to make primers used by DNA plymerase I that makes DNA & removes RNA primers
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RNAase H
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partially digests RNA leaving fragments that are primers for DNA polymerase I
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how to clone cDNA
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into plasmid or phage by attaching linker sequences to ends that are recognized by restriction enzyme to make sticky ends
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linker sequences?
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short double-stranded oligonucleotides containing restrction enzyme recognition sequence
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reverse transcriptase PCR (RT-PCR)
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reverse transcriptase generates single-stranded DNA copies of mRNA, product put in PCR to copy it into double-stranded molecules, then to amplify these copies - Taq polymerase & random primers added to single-stranded cDNA
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why use RT-PCR?
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more sensitive than conventional cDNA preparation & can identify mRNAs present in only one or two copies of cell
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probe
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labeled DNA or RNA sequence complementary to some part of a cloned sequence in the library - used to screen a library to recover clones of a specific gene
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why can clones carrying human ribosomal genes be recovered from a library using frog Xenopus laevis DNA probes?
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ribosomal gene sequences are highly conserved
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how to screen a plasmid library
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clones grown on nutrient agar plates; replica of the colonies made by pressing a nylon filter onto plate's surface - filter's bacteria then lysed, denatured & resulting single-stranded DNA bound to the folter to be screened with probe - double-stranded hybrid molecules will form for complementary strands & can be assayed PR if used radioactive probe can lay X-ray film on filter that will expose spots where useful colonies are
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plaque hybridization
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used to screen phage library
solution with recombinant phages spread over bacteria to form plaques that are transferred to a nylon membrane to bedenatured & probed |
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phage plaques are more efficient for use in screening large genomic libraries than plasmid colonies because
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plaques are much smaller so more can be screened on a single filter
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restriction map
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establishes the name, order & distance between restriction enzyme cleavage sites along a cloned segment of DNA - can serve as identity tags
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restriction map units
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bp - base pairs
kb - kilobase pairs |
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importance of restriction maps
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can define boundaries of a gene, dissect internal organization of a gene & locate mutations
used to map genes to specific human chromosomes & regions of chromosomes |
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if restriction enzyme recognition sequences is closely linked to a mutant allele that can be used as a marker in genetic carriers to
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identify carriers of recessively inherited disorders or prenatially diagnose a fetal genotype
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Southern blot method
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detects hybrids; used to identify which clones in a library contain a given DNA sequence & find sizes of chromosomes, or determine whether a clone contains all or only part of a gene
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2 components of southern blot technique
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separation of DNA fragments by gel electrophoresis & hybridization of fragments using labeled probes
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to make a southern blot DNA in the electrophoresis gel is denatured with ___ to ___.
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alkaline treatments to form single-stranded fragments
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to make a southern blot, transfer of DNA fragments to a membrane is achieved by
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placing the membrane & gel on a wick (sponge)in buffer sol'n - layers of blotting paper placed on he filter & weighted - capillary action of buffer through the gel
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in the southern blot, how is the filter prepared for hybridization
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placed in heat-sealed bag with a labeled single-stranded DNA probe for hybridization; excess probe washed away & hybridized fragments visualized on film
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northern blot
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like Southern blot but RNA is boud to a filter
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western blot
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procedure involving proteins bound to a filter
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use of northern blot
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info about expression of specific genes used to study paterns of gene expression in embryonic tissues, cancer & genetic disorders, detection of alternatively spliced mRNAs, measuring size of mRNA transcripts by comparison with a known marker RNA
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James Sanger & colleagues developed
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the most commonly used method of DNA sequencing that converts DNA to single strands used for synthesizing a series of complementary strands, each of which randomly terminates at a different, specific nucleotide. This produces a series of fragments that can be separated by electrophoresis & analyzed to figure out the sequence
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glyphosate
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herbicide effective at very [low] nontoxic for humans & rapidly degraded in soil - resistance transferred to crops like soybeans & maize - inhibits chloroplast enzyme EPSP synthase
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EPSP synthase
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enzyme inportant in amino acid synthesis - w'out it plants die - EPSP gene cloned from glyphosate-resistant E coli placed in plant leaves to grow resistant calluses formed by A tumifaciens
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three major GE crops in USA
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maize, soybeans & cotton
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golden rice
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nutritionally-enhanced rice - extra beta-carotene with 2 genes from daffodil & one from bacteria for use in Asia & Africa where vitamin A deficiency is rampant
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environmental risks of GE crops
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cross breeding with wild plants, invasiveness of the modified plant, loss of biodiversity
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ethics of GE crops
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possible allergies, cost for farmers as many GE crops don't produce viable seeds, labelling issues & who regulating bodies should be
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insulin production in bacteria
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original method of insulin production ; synthetic genes for A & B subjunits constructed by oligonucleotide syntesis & inserted adjacent to the lacZ gene (for beta-galactosidase) - produces fusion polypeptide with amino acid sequence for galactosidase attached to sequence coding for insulin subunit; these purified from bacterial extracts treated with cyanogen bromide that cleaves the fusion protein from the beat-galactosidase - the two subunits spontaneously unite when mixed to make an active insulin molecule
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conditions treatable by recombinant pharmaceuticals
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heart failure, hypertension
hemophilia cancer hepatitis diabetes |
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disadvantage to using prokaryotic hosts to synthesize eukaryotic proteins
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bacterial cells can't correctly process & modify many eukaryotic proteins; can't add sugars & phosphate groups needed for full activity
eukaryotic proteins produced in prokaryotic cells often don't fold properly |
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how to overcome difficulties involved in synthesizing eukaryotic proteins?
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second-generation methods
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second-generation methods
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use eukaryotic hosts - instead of grwoing in culture get proteins from milk of livestock
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second-generation methods in treatment of emphysema
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deficiency of alpha1-antitrypsin associated with the heritable form - gene coding for this protein cloned next to a sheep promoter sequence activating transcription in milk-producing cells - fusion gene injected into sheep zygotes in vitro, zygotes placed in surrogate dams - milk produced contains high []
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Dolly was loned to facilitate
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establishment of a flock of sheep that consistently produces high levels of human protein
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Pompe disease
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progressive & fatal autosomal recessive condition; metabolic disorder; children have poor muscle tone, oft. die before 2 from respiratory or cardiac complications
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how can Pompe disease be treated with recombinant tech
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treated with recombinant alpha-glucosidase produced in rabbit milk, now in clinical tests
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subunit vaccine
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consists of one of more surface proteins of the virus or bacterium - antigen stimulates production of antibodies against teh virus/bacterium
e.g. hepatitis B vaccine |
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2 types of commonly used vaccines
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inactivated (from dead samples) & attentuated (live that can't reproduce)
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production of the hepatitis B vaccine
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gene for hep B surface protein clined into YEC & produced in yeast host cellsm extracted & purified
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pro & con of subunit vaccine
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+:source of pure vaccine manufactured in controlled env't
-: developing countries have problems manufacturing, transporting 7 storing; most need refridgeration & injection under sterile conditions |
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prenatal diagnosis of sickle-cell anemia
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single amino acid substitution in beta-globin chain eliminates a cutting site for restriction enzymes MstII and CvnI & so changes pattern of fragments seen on Southern blts (large band vs. 2 smaller)- can also use to identify parental genotypes & genotypes of other family members who may carry gene - use fetal cells from CVS or amniocentesis; family members can be teted with blood sample
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MstII
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cuts 3 times in the region of normal beta-globin gene - to small fragments - in sickle cell the middle MstII site is eliminated so get one large fragment
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If a fetus has a large band and two small bands what is their genotype for sickle cell?
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Heterozygous carrier
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what percentage of all point mutations can be characterized by restriction enzyme analysis?
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5-10 percent
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what is necessary for synthetic oligonucleotides to be used as probes to detect mutant alleles?
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well-characterized mutant gene; mutated region has been sequences
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allele-specific oligonuclotides (ASO)
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synthetic prbes that can identify alleles that differ by as little as a single nucleotide
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ASOs vs. restriction enzymes
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ASOs detect single-nucleotide changes of all types including those that don't affect enzyme cutting sites
inreased resolution & wider application |
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sickle-cell anemia and other disorders can be screened for using
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a combination of ASOs and PCR analysis - this is rapid & highly accurate
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how to screen for sickle-cell anemia & other point mutations
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Beta-globin gene amplified by PCR, put on filter & hybridized to an ASO synthesized from a normal or mutant copy of the beta-globin gene - normal AA produces a dark spot (2 copies of allele), heterozygous a light spot (one copy), sickle-cell won't bind the probe so see nothing
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cystic fibrosis
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caused by deletion delta 508 in 70% of all mutant copies of the gene - defect in protin cystic fibrosis transmembrane conducance regulator which regulates chloride ion transport across the plasma membrane
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