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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Construction and Screening ofGene Libraries
Further Readings:
Genome II by TA Brown, Ch. 4;
Current Protocols in MolecularBiology by Ausubel et al., Ch. 5 and 6;
PNAS88:1731-35
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Tissue/Cell
mRNA
cDNA
(methylation; addition oflinkers, etc.)
DNA
Partial or
CompleteRestriction
Size Fractionation
Vectors
(cDNA: plasmids,
phageGenomic: phage, cosmid,BAC, PAC, YAC, TAC)
Restriction
Ligation
Transformation, in vitropackaging, etc
Screening for
desired clones
Amplify for long-
term storage
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Genomic Libraries from genomic DNA
frequency of hitsindependent of geneexpression levels
may contain promoters andintrons
cannot express inheterologous system
useful for genome analysis,map-based cloning,
promoter studies, etc
reverse transcription of mRNA
dependent
no promoters or introns
expression is feasible if linkedto a suitable promoter
useful for analysis of codingregions and gene functions
cDNA Libraries
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Representation and RandomnessQ = (1 1/n)N
P = 1 Q
P = 1 (1 1/n)N
(1 1/n)N = 1 P
ln (1 1/n)N = ln (1 P)
N = ln (1-P) / ln (1-1/n)
Q: probability of a clone not in arandom library
P: probability of including any DNAsequence in a random library
N: independent recombinant clones
to be screened
n: total number of clones needed tocover the total genome if the
clones are not overlapping (totalsize of genome divided by theaverage size of a single clonedfragment)
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Representation and RandomnessN = ln (1 P) / ln (1 1/n)
P: probability of includingany DNA sequence in arandom library
N: independent recombinantclones to be screened
n: total number of clonesneeded to cover the total
genome if the clones arenot overlapping (total sizeof genome divided by the
average size of a singlecloned fragment)
N = ln (1 P) / ln (1 m/T)
P: probability that each mRNAwill be represented once
N: independent recombinantclones to be screened
m: number of molecules of therarest mRNA in a cell
T: total number of mRNAmolecules in a cell
Genomic Libraries cDNA Libraries
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Representation and Randomness
N = ln (1P) / ln (11/n)
To have 99% chance ofgetting a desiredsequence, screen 4.6
times the total number ofbase pairs
N = ln (1P) / ln (1m/T)
Since it is hard to estimatedthe total number of mRNAmolecules and the number of
rarest mRNA molecules, it isdifficult to predict the exactnumber of clones to bescreened. In general, to haveat least one copy of everymRNA, 500,000 to 1,000,000independent cDNA clones
should be screened
Genomic Libraries cDNA Libraries
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Vectors for Genomic Libraries
Propagate in Saccharomycescerevisiae;
Three major elements:centromere for nuclear division;telomeres for marking the end ofthe chromosome; origins ofreplication for initiation of newDNA synthesis when the
chromosome divides Previously an important tool to
map complex genomes;
Problems: chimera, instability
(rearrangement)
230 - 1700 kb(length ofnatural yeast
chromosome)Average: 400-700 kb
YAC (yeastartificialchromosome)
RemarksInsert SizeVector
More details can be found here:
http://batzerlab.lsu.edu/Hum_Mol_Gen_Lectures/Hum%20Mol%20Genet%20Lecture%203%20Feburary%203,%202004.ppt
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Vectors for Genomic Libraries
A combination of BAC and P1 featuresSimilar to
BAC
PAC (P1-
derived artificialchromosome)
Plasmid vector containing the F factor replicon;
One copy per bacterial cell
Up to 300 kb
Average:
100 kb
BAC (bacterialartificial
chromosome)
With P1 plasmid replicon (single copy in E. coli) andRi plasmid replicon (single copy in Agrobacterium
tumefaciens) With T-DNA border and can transform plant directly
Similar to P1TAC(Transformable
artificialchromosome)
Deletion version of a natural phage genome
P1 phage genome is about 110 kb
Efficient packaging system
paccleavage site for recognition
P1 plamsid replicon and inducible P1 lytic replicon
loxPsite for Cre action
Maximumabout 100 kb
BacteriophageP1
RemarksInsert SizeVector
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Sources: www.genetics.wisc.edu/courses/spring04/466/files/bacteria2.pdf
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Sources: www.genetics.wisc.edu/courses/spring04/466/files/bacteria2.pdf
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pAD10SacBII
(30kb)
sacB
Ad2
pBR322 ori
P1 plasmidreplicon
pac
loxP
loxP
kan
P1 lyticreplicon
sacB
Sp6 T7
BamH 1Sfi 1 Not 1c 1 repressorbinding site
E.colipromoter
Sca 1
kan loxPloxP B BS S
pac
kan loxPloxP B BS S
pac
P1 vector for the construction ofP1 vector for the construction of
recombinants by P1 packagingrecombinants by P1 packaging
Pierce et al, Proc. Natl. Acad. Sci.U.S.A. (1992) 89, 2056-2060
Sources: http://batzerlab.lsu.edu/Hum_Mol_Gen_Lectures/Hum%20Mol%20Genet%20Lecture%203%20February%201 ,%202005.ppt
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Sources: http://batzerlab.lsu.edu/Hum_Mol_Gen_Lectures/Hum%20Mol%20Genet%20Lecture%203%20February%201 ,%202005.ppt
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Constructing P1 recombinants usingConstructing P1 recombinants usingpackaging extractspackaging extracts
S
pac
kan loxPB Skan loxPB Skan loxPB SloxP BloxP B
packaging
loxP B kan loxPB
insert of 80-90 kb
site-specific recombination
loxP
Sources: http://batzerlab.lsu.edu/Hum_Mol_Gen_Lectures/Hum%20Mol%20Genet%20Lecture%203%20February%201 ,%202005.ppt
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Electroporation Based System Large insert size
Low copy number origin for propagation
High copy origin for DNA production
Negative selection against non-recombinants
Very stable inserts
P1 Derived ArtificialP1 Derived ArtificialChromosomes (PACs)Chromosomes (PACs)
Sources: http://batzerlab.lsu.edu/Hum_Mol_Gen_Lectures/Hum%20Mol%20Genet%20Lecture%203%20February%201 ,%202005.ppt
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Vectors for Genomic Libraries
Genome size of phages is about 47 kb;
Packaging system is efficient and can
handle a total size of 78-105% of the genome;
Replacement vector system is usuallyemployed;
Pre-digested arms are commerciallyavailable for library constructions;
Useful for study of individual genes
Up to20-30kb
Phages
RemarksInsert
Size
Vector
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Vectors for Genomic Libraries
Contain F plasmid origin of
replication and cossite;
Low copy number and hence morestable
Similar tocosmid
Fosmids
Plasmid contain the cossite of phage and hence can use phagepackaging system;
Propagate in E. colias plasmids; Useful for subcloning of DNA inserts
from YAC, BAC, PAC, etc.
35-45 kbCosmid
RemarksInsertSize
Vector
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Vectors for cDNA Libraries
Maximum size for mRNA is about 8 kb, hence thecapacity of DNA insert is not a major concernhere;
Insertion vector system is usually employed; Useful for study of individual genes and their
putative functions Efficient packaging system, easy for gene transfer
into E. colicells, more representative thanplasmid libraries, subcloning and subsequentDNA manipulation processes are less convenientthan plasmid systems
Up to 20-30kb (for
replacementvectors) and10-15 kb (forinsertion
vectors)
Phages
Relatively easy to transform E. colicells although
may not be as efficient as the phage system forlarge scale gene transfer;
Less representative than phage libraries,subcloning and subsequent DNA manipulationprocesses are more convenient than the phagesystems
Up to 10-15kb
Bacterialplasmids
RemarksInsert SizeVector
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Combining the Advantage of the
Phage and Plasmid Systems
Embed a plasmid vector in a vector In vitropackage, transfect, propagate, and
screen as phages
Excise the plasmid for the vector andpropagate and manipulate as plasmidssubsequently, e.g.
Excise by filamentous helper phages (e.g.Strategene)
Excise by the Cre-loxsystem (Clontech)
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
ZAP Library (Stategene)
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
AAAAAAAA
mRNA
TTTTTTTTGAGCTC
+ Linker-primerReverse transcriptase (no RNase activities)
5-methyl dCTP, dATP, dGTP, dTTP
First Strand Synthesis
1st Strand
cDNACH3 CH3 CH3
linker-primer: reverse transcription, restriction site (XhoI)
5-methyl dCTP: protect internal sites
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
TTTTTTTTGAGCTC
+ RNase H and DNA polymerase I; dNTPs
CH3 CH3 CH3
2nd StrandcDNA
AAAAAAAACTCGAT
RNase H: remove mRNA in DNA-RNA hybrid
DNA Polymerase I: synthesis 2nd strand
Both enzymes added simultaneously
When RNase H starts to degrade the mRNA, residual RNAfragments may act as primers for initiation of DNA synthesis
DNA Polymerase 1 fills the gaps by nick translation
Second Strand Synthesis
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
TTTTTTTTGAGCTC
CH3 CH3 CH3
AAAAAAAACTCGAT
EcoRIAdapter and XhoI linker: directional cloning
Addition of Adapter
G...
AATTC...
... CTTAA
... G
+ EcoRI adapter; ligase
TTTTTTTTGAGCT
CH3 CH3 CH3
AAAAAAAAC
G...
AATTC...
+ XhoI
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Primary Library Synthesis Size fractionation Ligation to arms
In vitropackage (packaging extract should be McrA-,McrB-, and Mrr- to prevent digestion ofhemimethylated DNA)
Host for phage infection, e.g. XL1-Blue MRF:Restriction deficient (mcrA)183, (mcrBC-hsdSMR-mrr)173;
supEto allow propagation of helper filamentous phages(with amber mutation); recA- to prevent recombination
F plasmid:
M15lacZfor blue-white screening
lacIq to prevent uncontrolled expression of fusion protein
F pili to allow infection of helper filamentous phages
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
ZAP Library (Stategene)
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Excision of pBluescript Plasmid
First host XL1-Blue MRF: Fplasmid provides F pilifor f1 filamentous phage attachment; it is asuppressing strain so that f1 helper phages whichcarry an amber mutation can pack DNA
The ZAP: contains initiation and termination signalsequences for f1 packing flanking pBluescriptsequence
Second host: SOLR, a non-suppressing strain tostop helper phage propagation; a phage resistantstrain to prevent phage propagation
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
F
XL1-Blue MRF
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
F
Co-transfection of ZAP and fl help phage
XL1-Blue MRF
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
F
The ZAP phage clone replicates in the host cell
The fl helper phage protein nicks the fl replication initiation
site I on ZAP and replication continues until reaching the flreplication termination site T
XL1-Blue MRF
T I
BIO L M i l P d b D H Mi L
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
F
fl phage coats indiscriminately pack DNA moleculescontaining the fl replication origin; both fl genome
and pBluescript fragments are packed
XL1-Blue MRF
BIO4320 L t M t i l P d b D H Mi L
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
F
fl phages are secreted to thegrowth medium
XL1-Blue MRF
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
F
SOLR
fl phages infect a new host SOLR (R
, suppressor free)
F
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
F
SOLR
fl phages infect a new host SOLR (R
, suppressor free)
F
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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
F
SOLR
fl genome (contains an amber mutation) cannot propagate inthe suppressor free SOLR and pBluescript containing cells
can be selected by the ampicillin resistance marker
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BIO4320 Lecture Materials, Prepared by Dr. Hon Ming Lam
Screening for the Right Clones
By PCR or hybridization if DNA sequenceinformation of the target gene or homologousgenes, or the amino acid sequence of thetarget gene product is available
By hybridization if DNA fragments of thetarget gene or homologous genes areavailable
By functional assays, e.g.Yeast functional complementation
Microinjection
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BIO4320 Lecture Materials, Prepared by Dr. Hon Ming Lam
Screening by Hybridization Plaque/colony lifting
Making labeled probes
HybridizationPre-hybridization
Hybridization
Washing Detection
Radioactivity
LuminescenceFlorescence
Chemiluminescence
Rescuing clones and further analysis
Pl /C l Lif i
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Plaque/Colony Lifting
Nylon membrane
Pl /C l Lifti
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Master plate
Replica membrane
Hybridization
Labelednucleic acidprobes(radioactive or
non-radioactive)
Plaque/Colony Lifting
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Hybridizationbottle
32P
Labeled probes hybridize toDNA bound on membrane
Wash offunboundprobe
Place Bio-Max film ontohybridized membrane
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Hybridizationbottle
Develop film
Original master plate
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The Result of 2
nd
Round Screening
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Making Probes
Methods of DetectionRadioactive (e.g. P32, P33, S35)
Non-radioactive (e.g. biotinylation,horseradish peroxidase, Digoxigenin)
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Example of Non-Radioactive Probes
(from Boehringer Mannheim)
DIG (Digoxigenin)
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Example of Non-Radioactive Probes
(from Boehringer Mannheim)
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Making Probes Methods of Synthesis
DNA
Nick translation (E. colipolymerase I)
Random priming (Klenow)
Random labeling by PCR 5-end labeling (DNA kinase)
3-end labeling (T4 DNA polymerase,
terminal transferase, modified T7 DNApolymerase, Klenow, reversetranscriptase)
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Nick Translation
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Nick Translation
Nicks created by DNaseI
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Nick Translation
Gaps created by E. coliDNApolymerase I (5 to 3 exonuclease
activities)
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Nick Translation
Gaps filled by E. coliDNA polymerase Iin the presence of dNTPs and labeled
deoxynucleotides (5 to 3 polymeraseactivities, proofreading by 3 to 5
exonuclease activities)
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Nick Translation
Reaction repeats to give uniform labeling
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Random Priming
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Random Priming
Heat denaturing
+ Random primers (hexamers);+ Klenow, dNTPs, and labeleddeoxynucleotides
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Making Probes Methods of Synthesis
RNA
Random labeling by in vitrotranscription(DNA dependent RNA polymerase, e.g.Sp6, T3, T7 RNA polymerase)
3-end labeling (DNA independent RNApolymerase + ATP by polyA tail; RNAligase)
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
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Making Riboprobes
Promoter
Clone gene of interest under the control
of a strong, specific, and induciblepromoter (e.g. Sp6, T3, T7)
Gene of Interest
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Making Riboprobes
Cut with a restriction enzyme to generate
a blunt end or 5 overhang at the 3 end ofthe gene of interest
Promoter Gene of Interest
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Making Riboprobes
+ DNA-dependent RNA polymerase
corresponding to the promoter used, inthe presence of NTPs and labelednucleotides, to generate randomly labeled
cRNA probes
Promoter Gene of Interest
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
H b idi ti d W hi
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Hybridization and Washing Prehybridization
for reduction of noise to signal ratio
Hybridization
usually carried out in solutions of high ionicstrength to maximize annealing rate
20-25C below Tm
smaller volume the better: fast reassociation andless probes
to minimize background, hybridize for the shortesttime with minimum probes
Washing to remove non-specific binding
can control the stringency by altering temperatureor [Na+]
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Conditions for Hybridization and Washing
For cloning of a specific gene, use highstringency conditions for hybridization (hightemperature, with formamide) and washing(high temperature, low NaCl)
For cloning of homologous genes (ormembers of a gene family), use low
stringency conditions for hybridization (lowtemperature, without formamide) andwashing (low temperature, high NaCl)
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Factors Affecting Hybrid Stability
Negligible effect with probes >500 bpDuplex length
Tm is reduced by 1oC for each 1% ofmismatching
Mismatchedbase pairs
Each 1% of formamide reduces the Tm byabout 0.6oC for a DNA-DNA hybrid
DestabilizingAgents
AT base pairs are less stable than GC basepairs in aqueous solutions containing NaCl
BaseComposition
Tm increases 16.6oC each 10-fold increase inmonovalent cations (Na+) between 0.01 to 0.40
M NaCl
Ionic Strength
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Tm: Transition Mid-Point;
Melting Temperature
Temperature at which 50% of thenucleotides of DNA-DNA, DNA-
RNA or RNA-RNA duplex arehybridized.
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For Hybrids > 100 Nucleotides
DNA-DNA
Tm=81.5oC + 16.6log10[Na+] + 0.41(%G+C) -0.63(% formamide) - (600/l)
DNA-RNA
Tm=79.8oC + 18.5log10[Na+] + 0.58(%G+C) +
11.8(%G+C)2 - 0.5(% formamide) - (820/l)
RNA-RNATm=79.8oC + 18.5log10[Na
+] + 0.58(%G+C) +11.8(%G+C)2 - 0.35(% formamide) - (820/l)
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For Hybrids > 100 Nucleotides
l = the length of the hybrid in base pairs [Na+] between 0.01 M to 0.4 M
%G+C between 30% to 75%
Stability: RNA-RNA > RNA-DNA > DNA-DNA Tm of a double-stranded DNA decreases by 1-
1.5oC with every 1% decrease in homology
pH between 5-9; when outside this range,stability of DNA decreases drastically due toprotonation or deprotonation of the bases
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For Oligos 14-70 nucleotides
Tm = 81.5oC + 16.6(log10[Na+])
+ 0.41(%G+C) - (600/N)
N=number of nucleotides
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For Oligos < 18 nucleotides
Tm = (#A+T x 2) + (#G+C x 4)
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Factors Affecting Hybridization Rate
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g y
Repetitive sequences increase the rateProbecomplexity
Each 10% of mismatching reduces rate by a factor of twoMismatchedbase pairs
Directly proportional to duplex lengthDuplex length
50% formamide: no effect; other concentrations: reducedDestabilizingAgents
Optimal rate at 1.5 M Na+
IonicStrength
Little effectBaseComposition
Increase rate of membrane hybridization; 10% dextransulfate increases rate by factor of tenViscosity
Maximum rate: 20-25oC below Tm for DNA-DNA hybrids, 10-15oC below Tm for DNA-RNA hybrids
Temperature
Little effect between pH 5.0 to 9.0pH
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Screening for the Right Clones
By PCR or hybridization if DNA sequenceinformation of the target gene or homologousgenes, or the amino acid sequence of thetarget gene product is available
By hybridization if DNA fragments of thetarget gene or homologous genes areavailable
By functional assays, e.g.Yeast functional complementation
Microinjection
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Screening by Yeast Functional
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g y
Complementation
Saccharomyces cerevisiaeis a simpleeukaryotic system
Relatively easy to grow and perform
genetic manipulation
Mutants, expression vector,
transformation protocol, etc. areavailable
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Cloning by Yeast Functional Complementation
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Cloning by Yeast Functional Complementation
A yeast mutant which is defective
in a function of your interest
A library containing cDNAs of theorganism of interest is inserted in
a yeast vector;
Yeastexpressionpromoter
cDNAs express under an
inducible yeast expression promoter
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Cloning by Yeast Functional Complementation
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Yeast mutant is complementedwhen the transgene expression is induced
Cloning by Yeast Functional Complementation
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Designing a Suitable Vector
E. coli
replicationorigin
Yeast
replicationorigin
E. coli
selection
marker
Yeastselection marker
Multiple
cloning site
Yeast expressionpromoter
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
A Case Study: Cloning of Potassium Channels
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A Case Study: Cloning of Potassium Channels
A yeast mutant which cannot survive
on K+
-limiting medium
A plant cDNA library constructed ina yeast vector;
Yeastexpressionpromoter
Plant cDNAs express under an
inducible yeast expression promoter
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
A Case Study: Cloning of Potassium Channels
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7/31/2019 Genomic Library_Hongming Lam
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Survives in K+-limiting mediumwhen the transgene expression is induced
A Case Study: Cloning of Potassium Channels
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
A Case Study: Cloning of Potassium Channels
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7/31/2019 Genomic Library_Hongming Lam
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Mutant Transformant
Both survive on K+
-rich medium
Only the transformant surviveson K+-limiting medium
Transgene not
induced
Anderson et al., 1992
A Case Study: Cloning of Potassium Channels