dna technology chapter 20. plasmid use plasmids are good tools for dna technology can be isolated...
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DNA TechnologyDNA TechnologyChapter 20
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Plasmid UsePlasmid UsePlasmids are good tools for DNA
TechnologyCan be isolated from bacterial
cellsOften carry resistance genesIsolated genes of interest can be
inserted into the plasmidHow is this insertion done?Restriction endonucleases
(enzymes)
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Restriction EnzymesRestriction EnzymesWhere were restriction enzymes
first found?Bacterial cellsThey were used to protect bacteria
from intruding phage DNABacterial DNA is modified to protect
it from its own restriction enzymesRestriction enzymes often cut DNA
leaving “sticky ends”
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Restriction sites are the regions on the DNA that the restriction enzyme cuts.
Why are restriction sites palindromes?
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How are restriction enzymes
used in DNA technology?
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Cloning Genes of InterestCloning Genes of InterestHow can a biologist make large
amounts of a gene and thereby produce lots of protein products?
Clone the genes in recombinant plasmids
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Which method of bacterial genomic
alteration is exploited in
this process?
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Genomic Genomic LibrariesLibraries
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cDNAcDNAWhat is the problem with
inserting a human gene into a bacterial plasmid?
Introns are not spliced in prokaryotes
How can this problem be solved?Reverse Transcription of mRNA
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Why is cDNA
shorter than the original eukaryotic
DNA?
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ProbesProbesRadioactive probes can tag a
specific gene sequence within a mass of DNA
Probes are single stranded compliments to known sections of the DNA
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Gel electrophoresisGel electrophoresisHow does gel electrophoresis
work?Uses electric charge to separate
molecules based on their sizeWhat charge does DNA have?NegativeWhich sized fragments will move
furthest through the gel?Smallest ones
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Restriction Fragment Restriction Fragment AnalysisAnalysisGenetic markers are regions of
DNA that vary from person to person
Usually located on non-coding regions of the DNA
Using restriction enzymes and gel electrophoresis, DNA of different individuals can be analyzed and compared
Extract DNA and treat it with restriction enzymes
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The red triangles indicate where the enzyme cuts the DNA.
Procedure:The restriction enzyme is added to the DNA being analyzed and incubated for several hours, allowing the restriction enzyme to cut at its recognition sites. The DNA is then run through a gel, which separates the DNA fragments according to size. You can then visualize the size of the DNA fragments and assess whether or not the DNA was cut by the enzyme.
Gel with an uncut and cut samples of DNA. Note that the sizes of the cut DNA fragments add up to the size of the uncut DNA.
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How could you detect the differences between these 2 alleles?
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Using RFLP Analysis to Using RFLP Analysis to Detect Harmful AllelesDetect Harmful AllelesHarmful, disease causing alleles
usually have identical RFLP’s within a family
Once the known RFLP’s for the normal and disease causing alleles are known family members can be tested using Southern Blot analysis
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Southern Blot:Southern Blot:
Load the gel with the DNA to be tested. Markers serve as standards for determining
sizes of DNA fragments.
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Separated DNA’s are denatured while still in the agarose, by soaking the gel in a basic solution
Single stranded DNA’s are transferred to a nylon membrane by blotting
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A Radioactively labeled probe is added to the nylon membrane
The probe is either RNA or DNA that will compliment a specific bp sequence on the DNA
After unbound probes have been washed away only bound probes remain on the blot
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VNTRVNTRA VNTR is variable numbered
tandem repeatTandem repeats are interspersed
throughout the genomeDifferent VNTR’s can be detected
using southern blot technique
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One VNTR is inherited from One VNTR is inherited from each parenteach parentSouthern blot analysis usually
shows 2 different bands one inherited from each parent
How could an individual have one band for the VNTR?
He/She inherited the same sized VNTR from each parent
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Three different alleles for Three different alleles for this particular VNTRthis particular VNTR
What are the different
genotypes for these 6
individuals?
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Frequency of VNTR’sFrequency of VNTR’sFrequency of allele pattern at a single
VNTR has been established for specific sites within the genome
What is the probability of matching a 5 locus DNA profile, where each locus is:0.01, 0.02, 0.03, 0.06 and 0.10?
One in 27.8 million people will randomly match this profile
OJ’s profile was of 24 different loci and he matched all 24!
The odds were 1 in 10 billion
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Amplification of DNAAmplification of DNAWhat enzymes would be necessary for
DNA amplification?DNA polymerase and ligaseWhat else would be necessary for the
process to work?Primers and nucleotides!How was the original DNA initially
uncoiled and unwound?HeatWhy did the heat cause difficulty with
the procedure?
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Mapping the Entire Mapping the Entire GenomeGenomeGene linkage mapping uses
recombination frequencies to construct linkage maps of chromosome
Chromosome walking will identify sequential regions of the chromosome
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Chromo-some
Walking
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DNA Sequencing uses defective nucleotides to sequence the DNA.
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In Situ HybridizationIn Situ HybridizationDenatured DNA is placed on slideradioactive single stranded probe
is used to identify complementary DNA
Used to identify genes that different species have in common
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Microassays use in situ
hybridization technique to determine
which genes are actively
being expressed in
the tissue sample
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Other uses for the new Other uses for the new technology….technology….
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Gene Gene TherapyTherapy
Stem cells are the best
candidates for this therapy
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Pharmaceutical ProductsForensicsEnvironmentAgriculturePaleontology
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Determining PaternityDetermining PaternityWhich child cannot belong to this set of Which child cannot belong to this set of parents?parents?
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Which lane represents the Which lane represents the father?father?
3
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Rape Rape InvestigatioInvestigationnDid the suspect Did the suspect commit the commit the crime?crime?
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Supplemental Lab 6ASupplemental Lab 6AIn this lab we will transform E.
coli bacterial colonies with recombinant plasmid DNA
It will be your job to distinguish between bacterial colonies that have been transformed with the recombinant plasmids
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Procedure for the Procedure for the TransformationTransformation
Click here to find out more about the procedure
Predict results for your procedure!