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MOLECULAR BIOLOGY – Molecular biology techniques
MOLECULAR BIOLOGY TECHNIQUES I.
DNA isolation and fragmentationRecombinant DNAGel electrophoresisHybridizationSouthern blot, RFLP
MOLECULAR BIOLOGY – Molecular biology techniques
1866 1910
19441953
Mendel
Morgan
Watson & CrickAvery, MacLeod & McCarty
human DNA:3 200 000 000 letters
Approx 25,000 genes
filling 150x
MOLECULAR BIOLOGY – Molecular biology techniques
Human genome sequenceNOT BAD WORK
INSIDE 150 YEARS !!!!
1977
Sanger
2004
START STOP
A U G G C A A U C A A G U G C U A A
RNAT A C C G T T A G T T C A C G A T T
A T G G C A A T C A A G T G C T A A
GTTTATTGCATTCTTCTGTGAAAAGAAGCTGTTCACAGAATGATTCTGAAGAACCAACTTTGTCCTTAACTAGCTCTTTTGGGACAATTCTGAGGAAATGTTCTAGAAATGAAACATGTTCTAATAATACAGTAATCTCTCAGGATCTTGATTATAAAGAAGCAAAATGTAATAAGGAAAAACTACAGTTATTTATTACCCCAGAAGCTGATTCTCTGTCATGCCTGCAGGAAGGACAGTGTGAAAATGATCCAAAAAGCAAAAAAGTTTCAGATATAAAAGAAGAGGTCTTGGCTGCAGCATGTCACCCAGTACAACATTCAAAAGTGGAATACAGTGATACTGACTTTCAATCCCAGAAAAGTCTTTTATATGATCATGAAAATGCCAGCACTCTTATTTTAACTCCTACTTCCAAGGATGTTCTGTCAAACCTAGTCATGATTTCTAGAGGCAAAGAATCATACAAAATGTCAGACAAGCTCAAAGGTAACAATTATGAATCTGATGTTGAATTAACCAAAAATATTCCCATGGAAAAGAATCAAGATGTATGTGCTTTAAATGAAAATTATAAAAACGTTGAGCTGTTGCCACCTGAAAAATACATGAGAGTAGCATCACCTTCAAGAAAGGTACAATTCAACCAAAACACAAATCTAAGAGTAATCCAAAAAAATCAAGAAGAAACTACTTCAATTTCAAAAATAACTGTCAATCCAGACTCTGAAGAACTTTTCTCAGACAATGAGAATAATTTTGTCTTCCAAGTAGCTAATGAAAGGAATAATCTTGCTTTAGGAAATACTAAGGAACTTCATGAAACAGACTTGACTTGTGTAAACGAACCCATTTTCAAGAACTCTACCATGGTTTTATATGGAGACACAGGTGATAAACAAGCAACCCAAGTGTCAATTAAAAAAGATTTGGTTTATGTTCTTGCAGAGGAGAACAAAAATAGTGTAAAGCAGCATATAAAAATGACTCTAGGTCAAGATTTAAAATCGGACATCTCCTTGAATATAGATAAAATACCAGAAAAAAATAATGATTACATGAACAAATGGGCAGGACTCTTAGGTCCAATTTCAAATCACAGTTTTGGAGGTAGCTTCAGAACAGCTTCAAATAAGGAAATCAAGCTCTCTGAACATAACATTAAGAAGAGCAAAATGTTCTTCAAAGATATTGAAGAACAATATCCTACTAGTTTAGCTTGTGTTGAAATTGTAAATACCTTGGCATTAGATAATCAAAAGAAACTGAGCAAGCCTCAGTCAATTAATACTGTATCTGCACATTTACAGAGTAGTGTAGTTGTTTCTGATTGTAAAAATAGTCATATAACCCCTCAGATGTTATTTTCCAAGCAGGATTTTAATTCAAACCATAATTTAACACCTAGCCAAAAGGCAGAAATTACAGAACTTTCTACTATATTAGAAGAATCAGGAAGTCAGTTTGAATTTACTCAGTTTAGAAAACCAAGCTACATATTGCAGAAGAGTACATTTGAAGTGCCTGAAAACCAGATGACTATCTTAAAGACCACTTCTGAGGAATGCAGAGATGCTGATCTTCATGTCATAATGAATGCCCCATCGATTGGTCAGGTAGACAGCAGCAAGCAATTTGAAGGTACAGTTGAAATTAAACGGAAGTTTGCTGGCCTGTTGAAAAATGACTGTAACAAAAGTGCTTCTGGTTATTTAACAGATGAAAATGAAGTGGGGTTTAGGGGCTTTTATTCTGCTCATGGCACAAAACTGAATGTTTCTACTGAAGCTCTGCAAAAAGCTGTGAAACTGTTTAGTGATATTGAGAATATTAGTGAGGAAACTTCTGCAGAGGTACATCCAATAAGTTTATCTTCAAGTAAATGTCATGATTCTGTTGTTTCAATGTTTAAGATAGAAAATCATAATGATAAAACTGTAAGTGAAAAAAATAATAAATGCCAACTGATATTACAAAATAATATTGAAATGACTACTGGCACTTTTGTTGAAGAAATTACTGAAAATTACAAGAGAAATACTGAAAATGAAGATAACAAATATACTGCTGCCAGTAGAAATTCTCATAACTTAGAATTTGATGGCAGTGATTCAAGTAAAAATGATACTGTTTGTATTCATAAAGATGAAACGGACTTGCTATTTACTGATCAGCACAACATATGTCTTAAATTATCTGGCCAGTTTATGAAGGAGGGAAACACTCAGATTAAAGAAGATTTGTCAGATTTAACTTTTTTGGAAGTTGCGAAAGCTCAAG
How to study th
is amazing amount of in
formatio
n?
MOLECULAR BIOLOGY – Molecular biology techniques
MOLECULAR BIOLOGY – Molecular biology techniques
1 ISOLATION OF DNA
High MW Genomic DNA Isolation
Typical Procedure1 Cell Lysis
– 0.5% SDS + proteinase K (55oC several hours)
2 Phenol Extraction– gentle rocking several
hours
Phenol Extraction• mix sample with equal volume
of sat. phenol soln• retain aqueous phase• optional chloroform/isoamyl
alcohol extraction(s)
aqueous phase (nucleic acids)
phenolic phase (proteins)
MOLECULAR BIOLOGY – Molecular biology techniques
ORGANIC PHASE SEPARATION
High MW Genomic DNA Isolation
Typical Procedure1 Cell Lysis
– 0.5% SDS + proteinase K (55oC several hours)
2 Phenol Extraction– gentle rocking several
hours
3 Ethanol/ salt Precipitation
EtOH Precipitation• 2-2.5 volumes EtOH, -20oC• high salt, pH 5-5.5• centrifuge or ‘spool’ out
MOLECULAR BIOLOGY – Molecular biology techniques
4 RNAse followed by proteinase K
5 Repeat Phenol Extraction and EtOH ppt
PLASMID DNA
MOLECULAR BIOLOGY – Molecular biology techniques
Natural Bacterial Transformation/ conjugation
Also possible to experimentally ‘transform’ plasmid vectors into bacteria -
see laterS. Pneumoniae ‘transforming’ DNA is a
plasmid
PLASMID DNA ISOLATIONAlkaline lysis denaturation/ renaturation protocol
MOLECULAR BIOLOGY – Molecular biology techniques
Protein denaturation (SDS)Single stranded plasmid DNASingle stranded genomic DNA
Bacteria lysed in
SDS + strong NaOH buffer
DENATURATION
Small multi-copy plasmid DNA quickly re-anneals in solution
Large single copy genomic DNA fails to re-anneal and
forms precipitate with proteins
Potassium acetate
pH NEUTRALISATION
SEDIMENTATION
Centrifugation
Aqueous (double stranded plasmid
DNA)
Pellet (proteins and genomic
DNA)
MOLECULAR BIOLOGY – Molecular biology techniques
PLASMID DNA ISOLATIONAqueous (double stranded plasmid
DNA)
Pellet (proteins and genomic
DNA)
1. Phenol/ CHCl3 extraction & Ethanol/ Salt precipitation
or 2. Solid phase/ silica extraction ‘miniprep’
Quick relatively pure double stranded plasmid DNA
In presence of alkaline chaotropic salts, denatured plasmid DNA binds to silica
beads in the column
Wash buffers used to remove impurities &
DNA eluted (and re-natured in H2O)
Centrifugation steps
MOLECULAR BIOLOGY – Molecular biology techniques
PLASMID DNA ISOLATIONAqueous (double stranded plasmid
DNA)
Pellet (proteins and genomic
DNA)
or 3. Anion exchange column-based chromatography
Altering the pH and ionic conditions removes impurities leading to high [salt] elution and EtOH or isopropanol
precipitation
Extremley pure double stranded plasmid DNA
Isolation of RNASpecial Considerations
• RNAse inhibitors!• extraction in guanidine salts• phenol extractions at pH 5-6
• (pH 8 for DNA)• selective precipitation of high MW
forms (rRNA, mRNA) with LiCl• oligo-dT column for mRNA’s• treatment with RNase-free DNase
MOLECULAR BIOLOGY – Molecular biology techniques
Guanidinium thiocyanate
Using UV spectroscopy to analyze DNA/ RNAUsing UV spectroscopy to analyze DNA/ RNA• Nucleic acids absorbs UV light with a major peak at 260nm (max)
Abs
orb
ance
Wave Length ()260
MOLECULAR BIOLOGY – Molecular biology techniques
• Detection
• Quantitation
• Assessment of purity
• Absorbance extinction coefficients () vary depending on the nucleic acid structure
A260
Isolated
nucleotides
ss RNA/ DNA
= 25
ds DNA
= 20
• A260 / A280 ratio indicates sample purity
Pure RNA = 2.0
Pure DNA = 1.8
Beer-Lambert equationA = cl
So now we have isolated DNA ... but it is still too long to work with:
2 how to fragment it?
- mechanical shearing (no control)
or ...
MOLECULAR BIOLOGY – Molecular biology techniques
MOLECULAR SCISSORS - TYPE II RESTRICTION ENDONUCLEASES
Hamilton Othanel Smith 1968
cohesive ends
MOLECULAR BIOLOGY – Molecular biology techniques
SPECIFIC CUT SPECIFIC JOINING (LIGATION)
Ability to join two foreign pieces of DNA together
MOLECULAR BIOLOGY – Molecular biology techniques
BLUNT END
CO
HE
SIV
E E
ND
S
RECOMBINANT DNA TECHNOLGY
MOLECULAR BIOLOGY – Molecular biology techniques
The plasmid as DNA ‘vector’ (vehicle)
Possible to insert ‘interesting’ DNA’s into a plasmid using restriction endonucleases
3
RECOMBINANT DNA TECHNOLGY
MOLECULAR BIOLOGY – Molecular biology techniques
The plasmid as DNA ‘vector’ (vehicle)
The recombinant plasmid containing the ‘interesting’ DNA sequence can now be propagated/ amplified by experimentally transforming the recombinant plasmid
into bacteria and allowing these bacteria to multiply and produce more
recombinant plasmid
Specialized strains of bacteria can be permeablised by electroporation of heat shock
Therefore specific DNA fragments can be selectively propagated i.e. cloned
RECOMBINANT DNA TECHNOLGY
MOLECULAR BIOLOGY – Molecular biology techniques
Specialized plasmid cloning vectors
Muliple Cloning Site (MCS) contains many restriction sites to maximize target DNA
cloning potential
Plasmids contain genes that confer antibiotic resistance so that only successfully
transformed bacteria are propagated
3 Why not clone whole genomes?
MOLECULAR BIOLOGY – Molecular biology techniques
Each bacterial colony represents an amplified clone containing a recombinant plasmid
harbouring a distinct region of the genome
i.e. together they represent a ‘Genomic DNA Library’
Also possible to do this using cDNA copies of transcribed mRNAs resulting a ‘cDNA Gene Expression Library’
MOLECULAR BIOLOGY – Molecular biology techniques
Bacteriophage Lambdavectors
phage linear DNA genome
Non-essential region allowing that can be substituted by DNA to be cloned (approx 20Kb)
cos cos
Cosmids, phosmids, BACs and YACs to clone larger DNA fragments
DNA ISOLATION AMPLIFICATION
How to visualize DNA?
GEL ELECTROPHORESIS NUCLEIC ACID HYBRIDIZATION
size distinction sequence distinction
MOLECULAR BIOLOGY – Molecular biology techniques
4
+---
GEL ELECTROPHORESISfragmented DNA
MOLECULAR BIOLOGY – Molecular biology techniques
D-galactose 3,6-anhydroL-galactose n
agarose
Ethidium Bromide SYBR® Safeon blue light
MOLECULAR BIOLOGY – Molecular biology techniques
23 kb9,5 kb
Genomic DNA on gel:
MOLECULAR BIOLOGY – Molecular biology techniques
Plasmid DNA on gel:
MOLECULAR BIOLOGY – Molecular biology techniques
NUCLEIC ACID HYBRIDIZATION
Fluorescence In Situ Hybridization(FISH)
labeled probe
MOLECULAR BIOLOGY – Molecular biology techniques
Metaphase spread chromosomes on a slide
Biotin-11-dUTP
32P
radioactive labeling
Probe labeling by incorporation of modified (d)NTPs
AUTORADIOGRAPHY
Streptavidin
Yanti-DIGantibody
(DIG)
Fluorophores
conjugation
Enzymesalkaline phosphatasehorseradish peroxidase
chemiluminiscence
MOLECULAR BIOLOGY – Molecular biology techniques
DIG
DIG
DIG
DIG
DIG
DIG
DIG
DIG
DIG DIG
DIG
YHRPsubstrate
light YHRPsubstrate
light YHRPsubstrate
light YHRPsubstrate
lightYHRPsubstrate
light
YHR
Psubstrate
light
MOLECULAR BIOLOGY – Molecular biology techniques
DNA denaturation
Melting (denaturation) temperature depends on these major factors:- GC content (and therefore AT content)- sequence length- gaps in the annealed strands- salt concentration- pH- organic solvents (DMSO, formamide...)
GC rich
GC rich
AT richTemp Temp
MOLECULAR BIOLOGY – Molecular biology techniques
MOLECULAR BIOLOGY – Molecular biology techniques
CHROMOSOME PAINTING – MULTI COLOR FISH
MOLECULAR BIOLOGY – Molecular biology techniques
Particularly useful when diagnosing chromsomal abnormalities in certain forms of cancer (region specific barcoding on left and whole chromsosome paints on right)
Translocated chromsome segment
Where in organism is the gene expressed?
Detection of mRNA by in situ hybridization:
MOLECULAR BIOLOGY – Molecular biology techniques
Adaptation of DNA FISH protocol (removal of genomic DNA by predigestion with DNase and use of labeled RNA probes to detect expressed transcripts)
MOLECULAR BIOLOGY – Molecular biology techniques
How to analyze specific form of genes in genomic DNA?
e.g. successful intergration of a transgene into the genome of a transgeneic animal (mouse)
Figure 8-38 (part 1 of 4) Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Molecular biology techniques
Southern blot – transfer of DNA to membrane
fragmented DNA
e.g. fragmentation by restriction endonucleases
Figure 8-38 (part 2 of 4) Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Molecular biology techniques
Figure 8-38 (part 3 of 4) Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Molecular biology techniques
Labeled probe has sequence homology to DNA of interest e.g.
the hopefully integrated transgene
Figure 8-38 (part 4 of 4) Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Molecular biology techniques
e.g. bands reveal integrated transgenes
and size shows whether integration was correct
Figure 8-38 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Molecular biology techniques
SOUTHERN BLOTcombines DNA fragmentation, gel electrophoresis and hybridizationto analyze specific DNA sequences
Same procedure blotting RNA used to confirm gene mRNA expression called
NORTHERN BLOTTING
Similar principle used to blot proteins that are then detected by specific antibodies - WESTERN
BLOTTING
SINGLE NUCLEOTIDE POLYMORPHISM (SNP)
MOLECULAR BIOLOGY – Molecular biology techniques
Restriction Fragment Length Polymorphism (RFLP)
3’AGCTAGCGTGCTGTGATGTAGCTGATGCTGAATTCTGCGATGTT’55’TCGATCGCACGACACTACATCGACTACGACTTAAGACGCTACAA’3
3’AGCTAGCGTGCTGTGATGTAGCTGATGCTGAATGCTGCGATGTT’55’TCGATCGCACGACACTACATCGACTACGACTTACGACGCTACAA’3
SNP
EcoRI
3’AGCTAGCGTGCTGTGATGTAGCTGATGCTG AATTCTGCGATGTT’55’TCGATCGCACGACACTACATCGACTACGACTTAA GACGCTACAA’3
3’AGCTAGCGTGCTGTGATGTAGCTGATGCTGAATGCTGCGATGTT’55’TCGATCGCACGACACTACATCGACTACGACTTACGACGCTACAA’3
Restriction digestion by EcoRI
MOLECULAR BIOLOGY – Molecular biology techniques
RFLP
MOLECULAR BIOLOGY – Molecular biology techniques
Diagnosis of Genetic Diseases by RFLP
MOLECULAR BIOLOGY – Molecular biology techniques
Hybridization to filter
complementary
cloning
GENOMIC or cDNA EXPRESSION LIBRARY
MOLECULAR BIOLOGY – Molecular biology techniques
isolateDNA
.
genomic library
hybridisation
restrictiondigestion
MOLECULAR BIOLOGY – Molecular biology techniques
sub-clone
experimentation
How to see genes ...
... where in tissues?
... where on chromosomes?
How to see specificforms of genes?
How to clone and amplify genes?
southern blot
RFLP
in situ hybridization recombinant DNA
MOLECULAR BIOLOGY – Molecular biology techniques
genomic libraries
Table 8-3 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Molecular biology techniques