METHODS IN MOLECULAR BIOLOGY

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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 proteins in E. coli,
  expression vectors, fusion and tagged proteins.
• Antibodies, immunodetection methods. Analyses of
  gene expression at the protein level 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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Prehled transkripce a translace v eukaryotické
bunce
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Tvorba nukleových kyselin
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Single Stranded Nicks in DNA
Hydrolysis of this ester bond
H2O
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Restriction/Methylation Enzyme
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Eco RI Restriction Enzyme
Single stranded nick

• First restriction enzyme from Escherichia coli,
  so Eco R1

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Restriction Enzyme Recognition Sites
Restriction sites are general palindromic
Able was I, ere, I saw Elba
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Restriction Enzyme Recognition Sites
BglII 5 A-G-A-T-C-T T-C-T-A-G-A 5
Isoschizomers In certain cases, two or more
different enzymes may recognize identical sites.
(e.g. MboI also cleaves at GATC, and so is an
isochizomer of Sau3A.)
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Frequency of cutting of recognition enzymes
Sau 3A (GATC) cuts (¼)(¼)(¼)(¼) once every
256 base pairs (assuming G/C A/T, which is
often does not) BamH1 (GGATCC) cuts
(¼)(¼)(¼)(¼)(¼)(¼) once every 4Kb HindII
(GTPyPuAC) cuts (¼)(¼)(½)(½)(¼)(¼) once every
1Kb
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Sticky ends
5 overhang (EcoRI) 5-GAATTC-3 ?
5-G-OH PO4-AATTC-3
3-CTTAAG-5 3-CTTAA-PO4
HO-G-5

3 overhang (PstI) 5-CTGCAG-3 ?
5-CTGCA-OH PO4-G-3
3-GACGTC-5 3-G-PO4
HO-ACGTC-5

Blunt ends
5 overhang (SmaI) 5-CCCGGG-3 ?
5-CCC-OH PO4-GGG-3
3-GGGCCC-5 3-GGG-PO4
HO-CCC-5

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Ligation of compatible sticky ends
Human DNA cleaved with EcoRI Corn DNA
cleaved with EcoRI

PO4-A-A-T-T-C-A-G-C-T-A-C-G-3
HO-G-T-C-G-A-T-G-C-5
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Agarose Gel Electrophoresis
_
DNA is negatively charged from the phosphate
backbone
Agarose mesh

Visualize DNA with ethidium bromide fluoresces
orange ONLY when bound to DNA
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1 2 3 4
1
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1 2 3 4
1
10 kb
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1 2 3 4
1
10 kb
2
Eco R1
7 kb
3 kb
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1 2 3 4
1
10 kb
2
Eco R1
7 kb
3 kb
3
PstI
6 kb
4 kb
PstI
4 kb
6 kb
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Plasmid vectors

• Circular DNA molecules that replicate
  independently of E. coli chromosome.
• Are present in various copy per cell - Some are
  very high copy (can be gt 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 reporter for ease of selecting inserts)

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Useful Plasmid Features

• Relaxed Replication
• Selectable Markers
• Streamlined
• Polylinker or MCS
• Identification of Recombinants
• most derived from pUC or pBR322

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pBluescript
origin of replication
A widely used plasmid cloning vector
ampicillin resistance gene
MCS
MCS, Multiple Cloning Site
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Ligation Reaction

• mix foreign and vector DNA in presence of DNA
  ligase
• optimal ratios of vector to insert generally
  1.5-21
• intermolecular base-pairing can occur between
  compatible overhangs

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• Kinases and Phosphatases
• add or remove phosphate groups and the 5 ends of
  DNA or RNA.

Kinase ATP
PO4-GATC
HO-GATC
Phosphatase
gamma
alpha
beta
- 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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Purification of Plasmids
Takes advantage of distinct topological state of
plasmids. - plasmids will be covalently closed,
negatively wound circles when E. 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 strands following 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 enhance DNA 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
chromosome
E. Coli cell Amps
Ampr
Permeablize membrane with Ca2 and heat shock
Select for growth in the presence of ampicillin
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• select for transformants with antibiotic
• electroporation 109-1010 colonies/?g DNA
• heat-shock 105-109 colonies/?g DNA)

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Identifying Recombinants

• based on interruption of a gene
• eg., lacZ gene b-galactosidase
• intact b-galactosidase produces blue color in
  presence of X-gal
• ?-complementation or blue-white screening