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Quiz

BIOL 239 Quiz: Set 20- Genetic Analysis
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by OneClass1329300 , Winter 2017
7 Pages
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Department
Biology
Course Code
BIOL239
Professor
Christine Dupont
Study Guide
Quiz

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Genetic Analysis
Things to recall with a library?
oLibraries are used to track a specific fragment, but in order to determine which
cell has it use probes to bind to complimentary gene areas (probes are single
stranded)
oThey isolate fragments that wouldn’t normally be isolated as they are in a smear)
Describe probes in more detail
oShort single-stranded stretches of DNA of known composition, form 25 to several
thousand nucleotides in length
o1) To visualize them, the probes are labeled, usually with 32P or fluorescent dyes
Used in the identification of clones that contain complementary DNA sequences.
From 25 to several thousand nucleotides
in length
oSeveral ways to make labeled probes Can be
chemically synthesized, and are
termed
oligonucleotides
(usually 25 to 100
nucleotides in length).
Fluorescent dyes can be added as part of the
synthesis procedure.
oMust know the DNA sequence of the probe
o2) Can label a previously cloned fragment
Removal from the vector by restriction endonuclease
digestion, followed by chemical
labeling
usually 100 - 500 base pairs in
length
Don't need to know the sequence of this probe
Error! Filename not specified.
Describe screening through hybridization
oIsolating gene
Replica plating allows to find exact l
ocation on master plate
oNylon adheres to DNA, all junk can be washed
away
oNaOH used to break hydrogen bonds
and make DNA single stranded
oThis break allows probe to bind to
complementary gene
oLook where different on replica plate, see where is
on master plate to find gene of interest
Get mRNA for gene of interest --> Make it cDNA (single stranded) --> Get colony
library which has normal DNA and your inserted vector from before --> Replica to nylon
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--> Kill cells to expose DNA --> NaOH to make single --> add cDNA probes --> Wash
away non annealled stuff --> X ray to see where colony with vector of interest is located
--> Refer to master plate
Error! Filename not specified.
Evaluate screening through hybridization
oThe region of complementarity between probe and target sequence
must be long enough to allow sufficient hydrogen bonds to provide a
cohesive force
oIn general, two single DNA strands that are longer than 50-100
bp will hybridize so long as the extent of their complementarity
is more than 80%
needs to fit both parameters to work
Error! Filename not specified.
Describe Southern Blot Analysis
oUsed to show the presence of a gene within a larger fragment of DNA Involves
the transfer of DNA fragments from an agarose gel to nitrocellulose filter paper
oThe relative position of the DNA fragments is preserved in the transfer
Hybridization between a specific probe and DNA on the filter can be carried out
oNot isolating gene, just want to see if have it
oNylon membrane makes stick in place (not diffuse once electrogradient is
removed) render single stranded w/ NaOH
oCapillary action of buffer below goes up imprinting DNA to nylon membrane.
Paper towels for capillary action
oShows if an individual has a gene, if so how frequent is it?
Error! Filename not specified.
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find more resources at oneclass.com

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Description
GeneticAnalysis  Things to recall with a library? o Libraries are used to track a specific fragment, but in order to determine which cell has it use probes to bind to complimentary gene areas (probes are single stranded) o They isolate fragments that wouldn’t normally be isolated as they are in a smear)  Describe probes in more detail o Short single-stranded stretches of DNA of known composition, form 25 to several thousand nucleotides in length o 1) To visualize them, the probes are labeled, usually with 32P or fluorescent dyes Used in the identification of clones that contain complementary DNA sequences. From 25 to several thousand nucleotides in length o Several ways to make labeled probes Can be chemically synthesized, and are termed oligonucleotides (usually 25 to 100 nucleotides in length). Fluorescent dyes can be added as part of the synthesis procedure. o Must know the DNA sequence of the probe o 2) Can label a previously cloned fragment  Removal from the vector by restriction endonuclease digestion, followed by chemical labeling  usually 100 - 500 base pairs in length  Don't need to know the sequence of this probe  Error! Filename not specified.  Describe screening through hybridization o Isolating gene Replica plating allows to find exact l ocation on master plate o Nylon adheres to DNA, all junk can be washed away o NaOH used to break hydrogen bonds and make DNA single stranded o This break allows probe to bind to complementary gene o Look where different on replica plate, see where is on master plate to find gene of interest  Get mRNA for gene of interest --> Make it cDNA (single stranded) --> Get colony library which has normal DNA and your inserted vector from before --> Replica to nylon --> Kill cells to expose DNA --> NaOH to make single --> add cDNA probes --> Wash away non annealled stuff --> X ray to see where colony with vector of interest is located --> Refer to master plate  Error! Filename not specified.  Evaluate screening through hybridization o The region of complementarity between probe and target sequence must be long enough to allow sufficient hydrogen bonds to provide a cohesive force o In general, two single DNA strands that are longer than 50-100 bp will hybridize so long as the extent of their complementarity is more than 80% needs to fit both parameters to work  Error! Filename not specified.  Describe Southern Blot Analysis o Used to show the presence of a gene within a larger fragment of DNA Involves the transfer of DNA fragments from an agarose gel to nitrocellulose filter paper o The relative position of the DNA fragments is preserved in the transfer Hybridization between a specific probe and DNA on the filter can be carried out o Not isolating gene, just want to see if have it o Nylon membrane makes stick in place (not diffuse once electrogradient is removed) render single stranded w/ NaOH o Capillary action of buffer below goes up imprinting DNA to nylon membrane. Paper towels for capillary action o Shows if an individual has a gene, if so how frequent is it?  Error! Filename not specified.  Describe PCR o Artificial DNA xeroxing o With the use of 2 specific oligonucleotides (primers), any fragment of DNA can be amplified from 1 to ~ 1 billion copies in as little as 2 hr. o PCR primers need to be 100% correct, so need to know sequence o Has uses in DNA cloning, DNA sequencing, making DNA probes, identification of organisms w/o Southern blotting o Amplifies up gene region in between two primers. Design two primers that are complementary to each end of the sequence of interest. o Exponentially more DNA strands are made o Primers have their 3' facing target region o Mix the primers, template DNA, thermostable DNA polymerase (e.g. Taq) and thenucleotide bases in a microtube Begin thermocycling procedure  Error! Filename not specified.  Describe PCR cycles  Error! Filename not specified.  Why is PCR exponential?  Each cycle of PCR results in duplication of the strands, exponential  Error! Filename not specified.  Uses for PCR o Diagnosis of infection to see a gene exclusive to a specific bacteria o Paleomolecular biology to see what infectious disease they had back then  Describe DNA sequencing, Sanger sequencing? o Provides the highest resolution of a cloned DNA fragment: A complete description of its genetic information at the level of the nucleotide o Requires a
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