02. to 04. genetic material dna and probes

7/31/2019 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.

02. to 04. genetic material dna and probes

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