DNA Sequencing
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Transcript of DNA Sequencing
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DNA Sequencing
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E Pennisi Science 2012;337:1159-1161
Published by AAAS
ENCODE: ENCyclopedia Of DNA Elements
Objective:To identify all functional elements in the human genome sequence
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Zooming in.A diagram of DNA in ever-greater detail shows how ENCODE's various tests (gray boxes) translate DNA's features into functional elements along a chromosome.
E Pennisi Science 2012;337:1159-1161
Published by AAAS
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DNA Sequencing
Restriction enzymes (1973; Boyer & Cohen) cleave the polynucleotide to smaller fragments.These smaller fragments (100-200 base pairs) are sequenced.The two strands are separated.
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DNA Sequencing
Restriction enzymes cleave the polynucleotide to smaller fragments in specific patterns.
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DNA Sequencing
Single stranded DNA divided in four portions.Each tube contains adenosine, thymidine, guanosine, and cytidine plus the triphosphates of their 2'-deoxy analogs.
POCH2
OH
OO
O
OH
P
O
OH
P
O
HO base
HHO
O
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DNA Sequencing
The first tube also contains the 2,'3'-dideoxy analog of adenosine triphosphate (ddATP); the second tube the 2,'3'-dideoxy analog of thymidine triphosphate (ddTTP), the third contains ddGTP, and the fourth ddCTP.
POCH2
OH
OO
O
OH
P
O
OH
P
O
HO base
HH
O
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DNA Sequencing
Each tube also contains a "primer," a short section of the complementary DNA strand, labeled with radioactive phosphorus (32P).DNA synthesis takes place, producing a complementary strand of the DNA strand used as a template.DNA synthesis stops when a dideoxynucleotide is incorporated into the growing chain.
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DNA Sequencing
The contents of each tube are separated by electrophoresis and analyzed by autoradiography.There are four lanes on the electrophoresis gel.Each DNA fragment will be one nucleotide longer than the previous one.
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DNA Profiling
DNA sequencing involves determining the nucleotide sequence in DNA.The nucleotide sequence in regions of DNA that code for proteins varies little from one individual to another, because the proteins are the same.Most of the nucleotides in DNA are in "noncoding" regions and vary significantly among individuals.Enzymatic cleavage of DNA give a mixture of polynucleotides that can be separated by electrophoresis to give a "profile" characteristic of a single individual.
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The bloody glove?
T
TG
TGA
TGAC
TGACA
TGACAT
TGACATA
TGACATAC
TGACATACG
TGACATACGT
ddA ddT ddG ddCSequence of fragment
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A bloody glove?
T
TG
TGA
TGAC
TGACA
TGACAT
TGACATA
TGACATAC
TGACATACG
TGACATACGT
ddA ddT ddG ddC
A
AC
ACT
ACTG
ACTGT
ACTGTA
ACTGTAT
ACTGTATG
ACTGTATGC
ACTGTATGCA
Sequence of fragment
Sequence of original DNA
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Genetic Fingerprinting
OJ Simpson and the bloody glove!
ForensicsPaternityID-militaryFoodWineAnthropologyEvolution
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http://evolution.berkeley.edu/evolibrary/article/evo_01
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PCR: Polymerase Chain Reaction
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PCR
When a sample of DNA is too small to be sequenced or profiled, the polymerase chain reaction (PCR) is used to make copies ("amplify") portions of it.PCR amplifies DNA by repetitive cycles of the following steps.
1. Denaturation2. Annealing ("priming")3. Synthesis ("extension" or "elongation")
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PCR
Target region
(a) Consider double-stranded DNA containinga polynucleotide sequence (the target region)that you wish to amplify.
(b) Heating the DNA to about 95°C causes thestrands to separate. This is the denaturation step.
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(b) Heating the DNA to about 95°C causes thestrands to separate. This is the denaturation step.
(c) Cooling the sample to ~60°C causes oneprimer oligonucleotide to bind to one strand andthe other primer to the other strand. This is theannealing step.
PCR
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(c) Cooling the sample to ~60°C causes oneprimer oligonucleotide to bind to one strand andthe other primer to the other strand. This is theannealing step.
(d) In the presence of four DNA nucleotides andthe enzyme DNA polymerase, the primer is extended in its 3' direction. This is the synthesisstep and is carried out at 72°C.
PCR
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This completes one cycle of PCR.
(d) In the presence of four DNA nucleotides andthe enzyme DNA polymerase, the primer is extended in its 3' direction. This is the synthesisstep and is carried out at 72°C.
PCR
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This completes one cycle of PCR.
(e) The next cycle begins with the denaturationof the two DNA molecules shown. Both arethen primed as before.
PCR
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(e) The next cycle begins with the denaturationof the two DNA molecules shown. Both arethen primed as before.
(f) Elongation of the primed fragments completesthe second PCR cycle.
PCR
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(f) Elongation of the primed fragments completesthe second PCR cycle.
(g) Among the 8 DNAs formed in the secondcycle are two having the structure shown.
PCR
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The two contain only the target region andand are the ones that increase disproportionately in subsequent cycles.
(g) Among the 8 DNAs formed in the secondcycle are two having the structure shown.
PCR
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PCR
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Recombinant Methods
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Recombinant DNA : GMOs
Restriction enzymes (1973), plasmids, promoters, recombinant DNA (rDNA) -> New Organisms -> Genentech et. al. (1976)
http://www.nytimes.com/1999/12/07/business/robert-a-swanson-52-co-founder-of-genentech.html
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Transgenic CropsMonsanto Syngenta Luis?
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CLONING
Hello Dolly, and Lassie, and Tabby
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DNA Mutations
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Understanding Evolution : Personal response question
Mutation is a random process.a. agreeb. disagree
Ribozyme structure comes from Scott, W.G., Finch, J.T., Klug, A. (1995) The crystal structure of an all-RNA
hammerhead ribozyme: a proposed mechanism for RNA catalytic cleavage. Cell 81: 991-1002
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Substitution
Insertion
Deletion
Frameshift
DNA Mutations
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DNA Mutations
Enzymatic corrections are common
But, they are not always done
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DNA MutationsSickle Cell Anemia
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RNA
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E Pennisi Science 2012;337:1159-1161
Published by AAAS
ENCODE: ENCyclopedia Of DNA Elements
Objective:To identify all functional elements in the human genome sequence
![Page 37: DNA Sequencing](https://reader036.fdocuments.us/reader036/viewer/2022062813/5681649e550346895dd6850b/html5/thumbnails/37.jpg)
M Leslie Science 2013;339:25-27
Published by AAAS
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Epigenetics
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Epigenetics
http://learn.genetics.utah.edu/content/epigenetics/control/
• Chemical reactions switch parts of the genome off and on at strategic times and locations.
• Epigenetics is the study of these reactions and the factors that influence them.
• View video:http://learn.genetics.utah.edu/content/epigenetics/intro/
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Nucleosomes & Histones
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Nucleosomes & Histones
Neoplasia is characterized by "methylation imbalance"