02. to 04. Genetic Material DNA and Probes
Transcript of 02. to 04. Genetic Material DNA and Probes
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The Genetic material:
DNA
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The Central Dogma of Genetics
DNAtranscription Reverse
RNA
translation
transcr pt on
Protein
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A, T, G, C in DNA
A, U, G, C in RNA DNA is double stranded
as po ar y o
A T base pair, G C base pair
, ,generally referred as upstream and downstream.In RNA: A U base pair G C base pair. This type of
ase pa r ng n causes secon ary s ruc ure.
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1. 5 vs 3
2. Purines vs.Pyrimidines
3. A vs. G
4. C vs. T
5. Transitions vs.transversions
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applications
Genome composition andc aracter zat on
Implications in sequencing Primer design
PCR yields
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Polarity of
DNA
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Higher order organization of genomes
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Most chromosomal DNA does not code forroteins or RNAs:
. .,
25,000 genes x 2,000 bp per gene
5 x 107/3 x 109 = 1.67%
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o ecu ar ro es:
n i
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Concept of probes
For diagnostic tests, the agent that isused to detect the presence of a moleculein the sample.
A DNA sequence that is used to detectt e presence o a comp ementarysequence by hybridization with a nucleic
.
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Need for probes
Screen for the gene of interest Southern blot to understand enomic
structure and gene copy numbers
Northern blot for analysis of RNAexpression
Verification of allelic amplification in PCR
...
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a ng s pro es
5 end labeling Pol nucleotide Kinase
3 end labeling
Fill in a restriction enz me site
Terminal transferase labeling
Nick translation
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Size of the probe
v y
Yield of the probe
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Enzymatic reaction
Substrate
Environment (pH etc.)
Enz me
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5 end labeling
Substrate: DNA, -P*-ATP environment: buffer
Enzyme: polynucleotide kinase
Can label both double-stranded DNA andsingle-stranded DNA.
Good for oli onucleotide labelin
Can label RNA as well
Do not need tem late
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filling in restriction site
Substrate: DNA, dNTPs (with one of themlabeled)
Buffer
Enzyme: polymerase (T4 DNApolymerase, DNA polymerase I Klenowfragment; Taq polymerase
Template required
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filling in restriction site
DNA
Digest with EcoR1
TTAA
+ dT and dA*
AATT
TTAAA*A*TTAATT
TTA*A*
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transferase
Substrate: DNA, dNTPs (labeled) Buffer
Enzyme: terminal transferase
Tem late NOT re uired
(A*)n(A*)n
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Continuous labeling
Substrate: DNA + dNTPs Buffer
Making the new strand DNA with one (or
more) labeled dNTPs:1. Nick translation;
2. Random rimer labelin
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Nick translation:
Making nicks on double stranded DNAwith DNase I
DNA polymerase I has two majoract v t es: 5 to exonuc ease act v ty an5 to 3 polymerase activity
po ymerase ma ng e new s ranDNA with labeled dNTP while degrading
.
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Nick translation
s + ase
+ DNA polymerase I
S nthesizin 5 to 3 while de radin the ori inal strand
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translation
Concentration of DNase I
Temperature of incubation (16C)
Concentration of tem late DNA
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Random primer labeling method:
Heat denature DNA
Annealing of short random primers to ssDNA
Klenow polymerase makes the new strand
with labeled dNTP DNA synthesis continue until it reaches
the next primer.
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Random primer labeling
Heat denature
+ random short rimer
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primer labeling
Concentration of random primers Concentration of template DNA
Primer template radio
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Labeling PCR products
Label PCR primer
labeled.
Label 3 end b dATP at the end of PCR Label 5 end afetr PCR by kinase
Random primer
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Single-strand probes
DNA probes using phagemids RNA robes usin in vitro transcri tion:
T3 RNA polymerase
T7 RNA ol merase Sp6 RNA polymerase
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Oligonucleotide probes
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1. You know amino acid se uences,
need to obtain DNA sequences
2. Only a segment is conserved amonggenes in different species known todate, and you are interested in
isolating the gene from your speciesof interest.
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How long my probe should be?
Uniqueness of sequences:At each base osition, there are four
possibilities (A, C, G, T)
The probability for any given sequenceto exist in a genome is
1/4n
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Degeneracy of the genetic code
natural
64
codonsam noacids
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table
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Codons.html#Exceptions
Exceptions to the Code
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Exceptions to the CodeThe genetic code is almost universal. The same codons are assigned tothe same amino acids and to the same START and STOP si nals in thevast majority of genes in animals, plants, and microorganisms. However,some exceptions have been found. Most of these involve assigning oneor two of the three STOP codons to an amino acid instead.Mitochondrial genes
When mitochondrial mRNA from animals or microorganisms (but not fromplants) is placed in a test tube with the cytosolic protein-synthesizing
, , ,t r a n s l a t e d i n t o a p r o t e i n .The reason: these mitochondria use UGA to encode tryptophan (Trp)
.machinery, synthesis stops where Trp should have been inserted.In addition, most animal mitochondria use AUA for methionine notisoleucine and all vertebrate mitochondria use AGA and AGG as chain
terminators. Yeast mitochondria assign all codons beginning with CU tothreonine instead of leucine (which is still encoded by UUA and UUG as iti s i n c y t o s o l i c m R N A ) .Plant mitochondria use the universal code, and this has permittedangiosperms to transfer mitochondrial genes to their nucleus with greatease.
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Nuclear genesViolations of the universal code are far rarer for nuclear genes. A
ew un ce u ar eu aryo es ave een oun a use one or wo otheir three) STOP codons for amino acids instead.Nonstandard Amino Acids
listed above even though some of these may be chemically altered,e.g. by phosphorylation, at a later time. However, two cases have
is inserted by a tRNA into the growing polypeptide.Selenocysteine. This amino acid is encoded by UGA. UGA is still
used as a chain terminator, but the translation machinery is able todiscriminate when a UGA codon should be used for selenocysteinerather than STOP. This codon usage has been found in certainArchaea, eubacteria, and animals (humans synthesize 25 different
p r o t e i n s c o n t a i n i n g s e l e n i u m ) .pyrrolysine. In one gene found in a member of the Archaea, thisamino acid is encoded by UAG. How the translation machinerynows w en encoun ers w e er o nser a w
pyrrolysine or to stop translation is not yet known.
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Considerations for mixed oligo probes
# of total oligo in the probe = # ofdegeneracy. E.g.
Met-Gln-Ile-Gly-Leu = 15 mer
1 x 2 x 3 x 4 x 6 = 144 oligos
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Antibody probes
Antibodies recognize its protein antigen You can ex ress the anti en in
expression libraries
Screening a DNA expression library withantibodies
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How to select probes
You have the gene cloned: ds DNA
m n l n h n
Someone cloned the gene from cattle, butI am workin with i
There are only a short region that is
evolutionarily conserved: Oligonucleotideor Guessmers
I only know the protein sequence: Mixed
oligonucleotide or antibodies
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Reference books
Sambrook et al., 1989, Molecular cloning, a laboratory
manual:
Cha ter 10 Pre aration of radiolabelled DNA and RNAprobes
Chapter 11, Synthetic oligonucleotide probes
,antibodies and oligonucleotides
Cureent protocols in molecular biology, ed. Ausubel et al.,,
Nucleic acid hybridisation, ed. Hames and Higgins, IRLpress, 1986.