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8/14/2019 Methods in Molecular Biology List of Covered
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METHODS IN MOLECULAR BIOLOGY
List of covered topics:
Recombinant DNA, cloning vectors, gene libraries. Purification and labeling of nucleic acids, hybridization. DNA modifying enzymes, cloning strategies, DNA sequencing. PCR applications, degenerate, nested, touch-down PCR, RACE. Analyses of mRNA, S1 mapping and primer extension. Analyses of gene expression at the mRNA level: in situ hybridization, RT-PCR,
quantitative real-time RT-PCR, microarray technology.
Extraction and separation of proteins. Design and preparation of recombinant proteinsinE. coli, expression vectors, fusion and tagged proteins.
Antibodies, immunodetection methods. Analyses of gene expression at the proteinlevel: western blotting, immunocytochemistry.
Protein-protein interaction analyses: GST pull-down assays, immunoprecipitation,yeast two-hybrid system.
Protein-DNA interaction analyses: EMSA, DNase protection assays. Analyses of transcriptional regulation in cell transfection systems. Analyses of gene expression and function in vivo using transgenic methods in
eukaryotic models (Drosophila and C. elegans).
Open topic or discussion.
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Pehled transkripce a translace v eukaryotick buce
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Tvorba nukleovch kyselin
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Single Stranded Nicks in DNA
Hydrolysis of
this ester bond
H2O
P
O
O OH
O-
OH
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Restriction/Methylation Enzyme
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Eco RI Restriction Enzyme
First restriction enzyme fromEscherichia coli, so Eco R1
Single stranded nick
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Restriction Enzyme Recognition Sites
Restriction sites are general palindromic:
Able was I, ere, I saw Elba
Bam H1 site:5-GGATCC-3
3-CCTAGG-5
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Frequency of cutting of recognition enzymes
Sau 3A (GATC) cuts ()()()() = once every 256 base pairs
(assuming G/C = A/T, which is often does not)
BamH1 (GGATCC) cuts ()()()()()() = once every ~4Kb
HindII (GTPyPuAC) cuts ()()()()()() = once every ~1Kb
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5 overhang (EcoRI)
5-GAATTC-3 5-G-OH PO4-AATTC-3
3-CTTAAG-5 3-CTTAA-PO4
HO-G-5
Sticky ends
5 overhang (SmaI)
5-CCCGGG-3 5-CCC-OH PO4-GGG-3
3-GGGCCC-5 3-GGG-PO4
HO-CCC-5
Blunt ends
3 overhang (PstI)
5-CTGCAG-3 5-CTGCA-OH PO4-G-3
3-GACGTC-5 3-G-PO4 HO-ACGTC-5
+
+
+
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Human DNA cleaved with EcoRI Corn DNA cleaved withEcoRI
5-C-G-G-T-A-C-T-A-G-OH3-G-C-C-A-T-G-A-T-C-T-T-A-A-PO
4
PO4-A-A-T-T-C-A-G-C-T-A-C-G-3
HO-G-T-C-G-A-T-G-C-5
Ligation of compatible sticky ends
+
5-A-C-G-G-T-A-C-T-A-GA-A-T-T-C-A-G-C-T-A-C-G-3
3-T-G-C-C-A-T-G-A-T-C-T-T-A-AG-T-C-G-A-T-G-C-5
Complementary base pairing
+ DNA Ligase, + rATP
recombinant DNA molecule
5-A-C-G-G-T-A-C-T-A-G-A-A-T-T-C-A-G-C-T-A-C-G-3
3-T-G-C-C-A-T-G-A-T-C-T-T-A-A-G-T-C-G-A-T-G-C-5
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Agarose Gel Electrophoresis
_
+
DNA is negatively
charged from the
phosphate backbone
Visualize DNA with ethidium
bromide fluoresces orangeONLY when bound to DNA
Agarose mesh
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1 2 3 41
10 kb
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10 kb
1 2 3 41
Eco R1
3 kb 7 kb
2
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Eco R1
3 kb 7 kb
10 kb
1 2 3 41
2
PstI
4 kb 6 kb
4 kb6 kb
PstI
3
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Eco R1
3 kb 7 kb
10 kb
1 2 3 41
2
PstI
4 kb 6 kb
4 kb6 kb
PstI
3
6 kb
PstIEco R1
3 kb 1 kb
4
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Plasmid vectors
Circular DNA molecules that replicate independently ofE.coli chromosome.
Are present in various copy per cell - Some are very high copy
(can be > 100 per cell); Others are low copy (1-25 per cell).
Three key features of plasmid vectors:
1) Origin of replication (e. g. ColE1, very high copy 500
copies per cell).
2) Antibiotic resistance (or other selectable marker).
3) Multiple cloning site (often embedded in a LacZ reporterfor ease of selecting inserts)
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Useful Plasmid
FeaturesRelaxed ReplicationSelectable Markers
Streamlined
Polylinker or MCS Identification of
Recombinants
most derived from
pUC or pBR322
|SacI| |ScII| |XbaI||SpeI||BamH||SmaI||PstI||EcRI||EcRV||HIII||ClaI| |SalI||XhoI| |KpnI|
GAGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATACCGTCGACCTCGAGGGGGGGCCCGGTACC
CTCGAGGTGGCGCCACCGCCGGCGAGATCTTGATCACCTAGGGGGCCCGACGTCCTTAAGCTATAGTTCGAATAGCTATGGCAGCTGGAGCTCCCCCCCGGGCCATGG
Multiple Cloning Site:
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pBluescriptpBluescript
MCS
MCS,Multiple Cloning Site
ampicillin
resistancegene
A widely used plasmid
cloning vector
origin ofreplication
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mix foreign and vector DNA inpresence of DNA ligase optimal ratios of vector to insert generally
1.5-2:1
intermolecular base-pairing can occurbetween compatible overhangs
Ligation Reaction
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IV. Kinases and Phosphatases
add or remove phosphate groups and the 5 ends of
DNA or RNA.
HO-GATC PO4-GATC
Kinase +ATP
Phosphatase
O
A(P)-(P)-(P)-
- the enzyme is not sequence-specific
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Intramolecular vs. Intermolecular
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Removal of 5-PO4 Prevents
Vector Self Ligation
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TERMI ILO I G
REQUIRE M E NTS COM M E N T S
Identic lO er ngs
s t se trea tmen t f
linear lasmid im r esefficienc .
Restricti n sites at j ncti ns reser ed .
B t rien tati ns f insert DNA ssi le.Tandem c ies f insert ssi le.
Bl nt-endH igh c ncen trati ns f DNA
and ligaseneeded.hos ha tase trea tmen t.
Restriction sites at j nctions often
elimina ted . Tandem cop ies of insert DNApossi le. Bothorien tations possi le.
Different
O erhangs
rification ofdouble-cut
plasmid increases
efficienc .
Restriction sites at junctions preser ed .
Backgroundofnon-recombinants is low .
One possibleorien tation of insert. Tandemcop ies un likel .
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Purification of Plasmids
Takes advantage of distinct topological state of plasmids.
- plasmids will be covalently closed, negatively wound circles whenE. coli is
lysed.
- chormosomal DNA will be sheared into linear, non-topologically constrained
fragments (because so big).
This difference can be exploited to allow
purification of plasmids:
- difference in binding ethidium bromide,
leading to different densities (CsCl banding,
right).
- Different rater of re-associate of two strandsfollowing denaturation by boiling or alkaline
treatment
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Generic rDNA Protocol
prepare foreign DNA
prepare vector ligate foreign DNA and vector
introduce rDNA into host
heat-shock
electroporation
Transformation
incubate ligation mixture
with competent cells
cells pretreated to enhanceDNA uptake
treat according to method 40-41o for 1-2 minutes
brief pulse of high voltage
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Bacterial Transformation with a Plasmid
E. ColicellAmps
chromosome
Permeablize membranewith Ca2+ and heat shock
Ampr
E. ColicellAmpr+ plasmid
Select for growth in the presence of ampicillin
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Identifying Recombinants
based on interruption of a gene eg., lacZ gene = F-galactosidase
intact F-galactosidase produces
blue color in presence of -gal
E-complementation or blue-white screening