Download Lect9 Mol Biol Techniques

Molecular Biology
Techniques
Nicky Mulder
Acknowledgements: Anna Kramvis for lecture
material (adapted here)
Experiments for
different cell
processes
Two levels of experiment

Small-scale -1-10 genes/proteins:
 PCR
 Restriction
enzymes
 Cloning
 Hybridization

Large-scale 100-10000 genes or whole
genome -> High-throughput biology
Polymerase Chain Reaction
Fnlmh.ufl.edu/cowries/PCR
Agarose gel electrophoresis
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Agarose is used to form a gel
Gel is placed in solution with an anode and
cathode
DNA has net negative charge from sugarphosphate backbone –migrates towards anode
Migration speed is determined by size
Run DNA with some markers of known size
Visualized by ethidium bromide –flouresces in
UV light
Results on a gel
Restriction enzymes

Restriction enzymes recognize specific or
defined 4 to 8 base pair sequences on
DNA and cut
Microorganism
Enzyme
Sequences
Notes
Haemophilus
aegitius
HaeIII
5’…GG CC..3’
3’…CC GG..5’
Blunt
end
Haemophilus
haemolytica
HhaI
5’…GC G C..3’
3’…CG C G..5’
3’ single
strand
5’…G AATT C..3’
3’…C TTAA G..5’
5’ single
strand
Escherichia coli
EcoRI
Restriction maps
Restriction maps on gel
-
+
www.cbs.dtu.dk
Use of restriction enzymes

Cloning
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Restriction fragment length polymorphism
Restriction Fragment Length
Polymorphism (RFLP)
M1 M2
A2
A1 D
con
1.5 kb
1.0 kb
750 bp
671 bp
593 bp
500 bp
448 bp
400 bp
300 bp
200 bp
145 bp
StuI
uncut
Cloning
www.biodavidson.edu
Cloning vectors
Features:
Antibiotic resistance gene
Another marker gene
(lacZ*)
Specific promoter
Multiple cloning site
*Lac Z gene, encodes
beta-galactosidase- causes
bacteria expressing the
gene to appear blue when
grown on a medium that
containing X-gal
Other vectors
Bacterial artificial chromosomes
 Yeast artificial chromosomes
 Organism-specific vectors
 Expression vectors

Prokaryote gene transfer
Conjugation –transfer between bacteria by
direct contact
 Transduction –transfer of DNA via a virus
 Transformation –uptake of DNA from
environment by competent cells

Southern Hybridization
http://www.cdc.gov/ncidod/eid/vol6no1/images/vanderpoel1b.gif
Northern Hybridization
http://www.molecularstation.com/images/northern-blot-med.jpg
Western Hybridization
http://www.steve.gb.com/images/science/western_blotting.png
High-throughput biology
Move away from single gene focus and
bottom-up approach
 Studying multiple genes at once
 Using new technologies
 Moving from genotype to phenotype
 Trying to find function of sets of genes:
Functional genomics

Functional genomics experiments
DNA sequencing and analysis
 Mutagenesis and gene disruption
 DNA microarrays (transcriptomics)
 Proteomics (protein expression, 2D
gels, protein-protein interactions)
 Structural genomics
 Metabolomics
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Functional genomics & Bioinformatics
Large-scale experiments generating vast
amounts of data
 Data needs sorting and analysis
 Bioinformatics allows:
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 Tracking
of samples
 Automating data capture
 Data storage and analysis
 Data mining to convert data into biological
research
DNA sequencing technologies
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Sanger sequencing method (chain termination)
 Dideoxynucleotide
triphosphates (ddG/A/T/C/TP, lack
3-OH), labelled primers and DNA polymerase -4
reactions –run on gel
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Dye terminator sequencing
terminators with diff dyes –single reaction, use
capillary electrophoresis
 Label

High-throughput sequencing
reactions, DNA on surfaces –sequencing by
synthesis and detection of fluorescence
 Parallel
Sanger
sequencing
method
www.bio.davidson.edu/Courses/Bio111/dnaseq2.gif
Automated sequencing
Automated sequencing
Genome sequencing
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To sequence a fragment of DNA:
 subclone
fragment into vector- plasmid (2kb),
cosmid (40kb), BAC (>100kb) or YAC (1Mb)
 Grow cells and purify DNA
 Sequence user flourescent dye labels and
laser detection –can get 300-800bp per read
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Problem is if fragment is too big –not
covered by reads
Whole genome shotgun
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Need to fragment the DNA, sequence the
pieces and then assemble them
Need to over-sample to get good overlaps
May still get gaps using this approach, but
can design new primers for additional
sequencing
Repeats are an issue –can cause incorrect
assembly
Shotgun sequencing works for small
genomes like bacterial genomes
Sequencing complex genomes
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As the complexity increases so does likelihood of
incorrect assembly –eukaryotes has many repeats
Genome maps are important and form a guide for
showing positions of genes and features
Eukaryotic genomes are fragmented into 1.5Mb bits
and cloned into BACs, then a shotgun approach is
used for each BAC –hierarchical shotgun sequencing
These “contigs” are assembled as before, and
mapped onto genome using markers (genetic map)
Hierarchical shotgun sequencing
International
Human Genome
Sequencing
Consortium, 2001,
Nature 409, pg
860-921.
Assembly
Hierarchical
shotgun
sequencing
PHRED Base
calling, trace
quality,
Crossmatch –
finds vector
PHRAP Assembly:
Align fragments,
consensus quality
Highest quality
reads used for
consensus
CONSED
sequence
editing
Genome Annotation

Two main levels:
Annotation – Finding genes
and other biologically relevant sites thus
building up a model of genome as objects
with specific locations
 Functional annotation – Objects are used
in database searches (and expts) aim is
attributing biologically relevant information
to whole sequence and individual objects
 Structural
Genome structure
Gene prediction
Promoter
prediction
Translation
BLAST
Signatures
2D structure
3D structure
Genes, pseudogenes, introns,
exons, intergenic regions
Proteins
Functional annotation
Annotation can be at different levels
Function,
structure
Gene
regulation
Interactions,
pathways
Cellular process,
localisation
Gene expression -Transcriptomics
 Microarrays
 ChIP
on chip (Chromatin IP on
microarrays)
Microarray overview
Slide with target deposited
label cDNA (probe)
hybridise labelled probe to slide
wash slides
scan
analyse results
Microarray data analysis
Experimental design
Image processing
Normalization
Pre-processing
Data analysis
Data mining
Co-regulated genes have correlated
expression patterns
Data mining
Add gene identifiers
Add gene descriptions
Add GO terms
-GO0003456
-GO0006783
-GO0142291
-GO0054198
-GO0000234
-RNA polymerase
-Glycosyl hydrolase
-Phosphofructokinase
-Transcription factor
-Glucose transporter
Map onto pathways
-AB02387
-SB07593
-AA00498
-AC008742
-AB083121
Proteomics
Large-scale study of proteins to determine
their function
 Proteome is protein complement of the
genome
 Includes the study of:
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 Protein
structure and function
 Protein-protein interactions
 Protein expression
 Protein localization
 Protein modifications
Proteomics studies
Mass spectrometry
Xray, NMR
Mass spectrometry
Localization studies
Workflow of a proteomics experiment
Sample preparation
Protein separation
Protein selection
Protein identification
Sample can be from patient
cohort, cell selection, fraction,
etc.
Different separation
techniques, e.g. 2-D PAGE,
HPLC, ICAT, etc.
Depends on separation method
Usually mass spectrometry
Protein separation
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2D PAGE
Gel-free
systems:
 ICAT
 HPLC
Mass spec –digest proteins further
Size gradient
Protein separation -2D PAGE
pH gradient
Bioinformatics component
 Sample
tracking
 Image capture
 Image analysis and comparison:
Measuring
intensities
Removing background noise
Finding difference between gels
After 2D PAGE
Mass spectometry
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Digest proteins with e.g. trypsin (lysine or
arginine)
Proteins ionized and brought into gas phase
Move through mass analyzer which separates
them based on mass
Detector records presence of ions
Protein identification (MS)
Peptide Fragment Fingerprinting (PFF)
MS/MS or Tandem MS
Peptide identification (MS/MS)
VHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGL
AHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHK
digest with trypsin
Recognises lysine (K) & arginine (R)
denature
VHLTPEEK
SAVTALWGK
VNVDEVGGEALGR
LLVVYPWTQR
FFESFGDLSTPDAVMGNPK
VK
AHGK
K
VLGAFSDGLAHLDNLK
GTFATLSELHCDK
LHVDPENFR
LLGNVLVCVLAHHFGK
EFTPPVQAAYQK
VVAGVANALAHK
Mass spec
V
HLTPEEK
VH
LTPEEK
VHL
TPEEK
VHLT
PEEK
VHLTP
EEK
VHLTPE
EK
VHLTPEE
K
mass spectrum
compare with theoretical peptide spectra;
ID = best similarity
Summary
Aim of molecular biology experiments is to
understand biology
 Find gene/protein functions
 See what is causing a phenotype
 see if/when a gene or protein is expressed
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Cloning exercise