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MOLECULAR BIOLOGY Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization Southern blot, RFLP

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Page 1: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

MOLECULAR BIOLOGY – Molecular biology techniques

MOLECULAR BIOLOGY TECHNIQUES I.

DNA isolation and fragmentationRecombinant DNAGel electrophoresisHybridizationSouthern blot, RFLP

Page 2: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

MOLECULAR BIOLOGY – Molecular biology techniques

1866 1910

19441953

Mendel

Morgan

Watson & CrickAvery, MacLeod & McCarty

Page 3: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 4: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 5: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

MOLECULAR BIOLOGY – Molecular biology techniques

1 ISOLATION OF DNA

Page 6: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 7: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 8: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 9: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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)

Page 10: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 11: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 12: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 13: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 14: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 15: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 16: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

MOLECULAR BIOLOGY – Molecular biology techniques

BLUNT END

CO

HE

SIV

E E

ND

S

Page 17: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 18: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 19: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 20: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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’

Page 21: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 22: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

DNA ISOLATION AMPLIFICATION

How to visualize DNA?

GEL ELECTROPHORESIS NUCLEIC ACID HYBRIDIZATION

size distinction sequence distinction

MOLECULAR BIOLOGY – Molecular biology techniques

4

Page 23: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

+---

GEL ELECTROPHORESISfragmented DNA

MOLECULAR BIOLOGY – Molecular biology techniques

D-galactose 3,6-anhydroL-galactose n

agarose

Page 24: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

Ethidium Bromide SYBR® Safeon blue light

MOLECULAR BIOLOGY – Molecular biology techniques

Page 25: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

23 kb9,5 kb

Genomic DNA on gel:

MOLECULAR BIOLOGY – Molecular biology techniques

Page 26: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

Plasmid DNA on gel:

MOLECULAR BIOLOGY – Molecular biology techniques

Page 27: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

NUCLEIC ACID HYBRIDIZATION

Fluorescence In Situ Hybridization(FISH)

labeled probe

MOLECULAR BIOLOGY – Molecular biology techniques

Metaphase spread chromosomes on a slide

Page 28: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 29: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 30: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 31: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

MOLECULAR BIOLOGY – Molecular biology techniques

Page 32: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 33: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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)

Page 34: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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)

Page 35: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 36: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

Figure 8-38 (part 2 of 4) Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Molecular biology techniques

Page 37: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 38: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 39: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 40: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

SINGLE NUCLEOTIDE POLYMORPHISM (SNP)

MOLECULAR BIOLOGY – Molecular biology techniques

Page 41: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 42: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

RFLP

MOLECULAR BIOLOGY – Molecular biology techniques

Page 43: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

Diagnosis of Genetic Diseases by RFLP

MOLECULAR BIOLOGY – Molecular biology techniques

Page 44: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

Hybridization to filter

complementary

cloning

GENOMIC or cDNA EXPRESSION LIBRARY

MOLECULAR BIOLOGY – Molecular biology techniques

isolateDNA

Page 45: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

.

genomic library

hybridisation

restrictiondigestion

MOLECULAR BIOLOGY – Molecular biology techniques

sub-clone

experimentation

Page 46: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

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

Page 47: MOLECULAR BIOLOGY – Molecular biology techniques MOLECULAR BIOLOGY TECHNIQUES I. DNA isolation and fragmentation Recombinant DNA Gel electrophoresis Hybridization

Table 8-3 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Molecular biology techniques