1. Genetic Research “Big Picture” of Genetic Research Restriction Enzymes
Properties
Discovery
Use in genetic research
Polymerase Chain Reactions (PCR)
Principles
Use as modern tool in genetic research
Visualization of DNA using Agarose Gel Electrophoresis

2. Genomic Studies Complex pattern of gene expression Sequencing of
differential on/off of large number of genes
Sequencing of
Human Genome (2003, 13yrs)
30, genes
3.1billion bp
Post tranaslational mechanisms also contribute to this diversity and complexity

3. Genomic Studies Complex pattern of gene expression Sequencing of
differential on/off of large number of genes
Sequencing of
Human Genome (2003, 13yrs)
30, genes
3.1billion bp
Drosophila (14 000) Nematode (20 000)
Post tranaslational mechanisms also contribute to this diversity and complexity

4. Genomic Studies and/or chemical modification Alternative splicing
modulating gene expression
Post-transcriptional
mechanisms
Complex pattern of gene expression
differential on/off of large number of genes
and/or chemical
modification
Sequencing of
Human Genome (2003, 13yrs)
30, genes
3.1billion bp
Proteomic Studies
500,000-1 million proteins
40-60 % of genes alternatively spliced
1 gene = Avg of 3-4 isoforms
5% of genes alternatively spliced
Drosophila (14 000) Nematode (20 000)
Post tranaslational mechanisms also contribute to this diversity and complexity

5. Genomic Studies
Alternative splicing
modulating gene expression
Post-transcriptional
mechanisms
Complex pattern of gene expression
differential on/off of large number of genes
and/or chemical
modification
Sequencing of
Human Genome (2003, 13yrs)
30, genes
3.1billion bp
Proteomic Studies
300,000-1 million proteins
40-60 % of genes alternatively spliced
1 gene = Avg of 3-4 isoforms
5% of genes alternatively spliced
Drosophila (14 000) Nematode (20 000)
Purpose is to define meaningful knowledge of genome sequence
Identify When, Where, Why and How genes are expressed
50-60% of discovered human genes have unknown functions
Post tranaslational mechanisms also contribute to this diversity and complexity

6. Genomic Studies
Alternative splicing
modulating gene expression
Post-transcriptional
mechanisms
Complex pattern of gene expression
differential on/off of large number of genes
and/or chemical
modification
Sequencing of
Human Genome (2003, 13yrs)
30, genes
3.1billion bp
Proteomic Studies
300,000-1 million proteins
40-60 % of genes alternatively spliced
1 gene = Avg of 3-4 isoforms
5% of genes alternatively spliced
Drosophila (14 000) Nematode (20 000)
Purpose is to define meaningful knowledge of genome sequence
Identify When, Where, Why and How genes are expressed
50-60% of discovered human genes have unknown functions
FUNCTIONAL GENOMICS
Post tranaslational mechanisms also contribute to this diversity and complexity
Transcriptomics
Proteomics
Structural genomics
Comparative genomics

7. Genomic Studies How proteins interact
Alternative splicing
modulating gene expression
Post-transcriptional
mechanisms
Complex pattern of gene expression
differential on/off of large number of genes
and/or chemical
modification
Sequencing of
Human Genome (2003, 13yrs)
30, genes
3.1billion bp
Proteomic Studies
300,000-1 million proteins
40-60 % of genes alternatively spliced
1 gene = Avg of 3-4 isoforms
5% of genes alternatively spliced
Drosophila (14 000) Nematode (20 000)
Purpose is to define meaningful knowledge of genome sequence
Identify When, Where, Why and How genes are expressed
50-60% of discovered human genes have unknown functions
FUNCTIONAL GENOMICS
Post tranaslational mechanisms also contribute to this diversity and complexity
How proteins interact
with each other and the environment
Transcriptomics
Proteomics
Structural genomics
Comparative genomics
Normal
Abnormal

8. Genome-wide assessment of mRNA transcripts
Microarray Studies
Genome-wide assessment of mRNA transcripts
Whole process is based on hybridization of probe to target DNA
GREEN: Control DNA, RED:Experimental DNA
Microarrays contain up to 30,000 target spots
Microarray type
Application
Comparative Genomic Hybridization
Tumor classification, risk assessment, and prognosis prediction
Expression analysis (transcriptomics)
Drug development, drug response, and therapy development
Mutation/Polymorphism analysis
Drug development, therapy development, and tracking disease progression

9. SIGNALING NETWORK FOR EGFR FAMILY1 2 3 4 X X X P- -P -P
Shc
Grb2
p85
GAP
Gab1
SHP-1
Sos
p110
Src
Grb7
PLC-γ
Crk
STAT
Cbl
Raf
Eps
Ras
Dok-R
MEK
Akt
Nck
PKC
Ca++
Erk
CaMK
JNK
FOX
Sp1
c-jun
c-myc
NF-κB
c-fos
Elk
Ets
Stat
Transcription
PROLIFERATION
ANGIOGENESIS
DIFFERENTIATION
SURVIVAL
MIGRATION
ADHESION
Transgenic
Knockouts/Knockins
Condidional Expression
Adapted f rom Holbro et al. Ann Rev Pharmacol Toxicol., 2004, p195.

10. TESTING THERAPY IN ANIMALS
Shc
Grb2
p85
GAP
Gab1
SHP-1
Sos
p110
Src
Grb7
PLC-γ
Crk
STAT
Cbl
Raf
Eps
Ras
Dok-R
MEK
Akt
Nck
PKC
Ca++
Erk
CaMK
JNK
FOX
Sp1
c-jun
c-myc
NF-κB
TCR
c-fos
Elk
Ets
Stat

11. TESTING THERAPY IN ANIMALS
BASIC SCIENCE
CLINICAL TRIALS
INTERVENTION
THERAPY
TESTING THERAPY IN ANIMALS
OUTCOME
ELUCIDATION OF
MECHANISMS
PATIENT SAMPLES
SURVIVAL
Ras
MEK
Akt
Nck
PKC
Ca++
Dok-R
HUMAN
CELL LINES
Erk
CaMK
JNK
BILOGICAL
ASSOCIATIONS
FOX
Sp1
c-jun
c-myc
NF-κB
c-fos
Elk
Ets
TCR
Stat

12. Early 1970s: Genomic Studies: How to cut DNA into manageable
fragments? Chemical/mechanical means non-specific, non-reproducible
Breakthrough needed
Restriction Endonucleases: Molecular Scissors for Cutting DNA
Recognition sequence (6 bp)
Binding of endonuclease
to recognition site
Cutting of DNA producing
“overhanging”/“sticky”ends
(Can also produce blunt ends)

14. Restriction enzymes are usually homodimers

15. Restriction Enzymes Recognize Palindromic Sequences?

16. Restriction Enzymes Recognize Palindromic Sequences (Inverted Repeats)
DNA Sequence
Recognized
EcoRI
5'GAATTC 3'CTTAAG
BamHI
5'GGATCC 3'CCTAGG
                             
HindIII
5'AAGCTT 3'TTCGAA
MstII
5'CCTNAGG 3'GGANTCC
                               
TaqI
5'TCGA 3'AGCT
NotI
5'GCGGCCGC 3'CGCCGGCG
AluI
5'AGCT 3'TCGA

17. Restriction enzymes can be used to create a “MAP” of DNA
Cleavage of DNA with restriction enzymes provides landmarks and sequence information.
? MAP ?

18. Restriction enzymes can be used to create a “MAP” of DNA
Restriction enzymes played
important role in cloning of the
Human Genome
Digestion of DNA with restriction
Enzymes, cloning into vectors
Construction of restriction maps
of individual clones
Overlapping clones identified
by use of computers
Overlapping clones arranged
into contigs

19. FORMATION OF RECOMBINANT DNA

20. How were restriction enzymes discovered?

21. DISCOVERY FROM BACTERIAL CELLS

22. How is bacterial DNA protected from
restriction enzyme digestion?

23. Bacterial methylase adds methyl group to one or 2 base pairs
leading to a restriction-modification system
Example: In some bacteria, cytosine nucleotide has an extra
single carbon group added to it.
Restriction endonucleases no longer recognize the methylated DNA
and hence are protected from digestion.
This is how restriction enzymes got their names: They restrict the
synthesis of foreign DNA

24. Organism from which derived Target sequence (cut at *) 5' -->3'
Restriction enzymes recognize palindrome sequences
Enzyme
Organism from which derived
Target sequence (cut at *) 5' -->3'
Ava I
Anabaena variabilis
C* C/T C G A/G G
Bam HI
Bacillus amyloliquefaciens
G* G A T C C
Bgl II
Bacillus globigii
A* G A T C T
Eco RI
Escherichia coli RY 13
G* A A T T C
Eco RII
Escherichia coli R245
* C C A/T G G
Hae III
Haemophilus aegyptius
G G * C C
Hha I
Haemophilus haemolyticus
G C G * C
Hind III
Haemophilus inflenzae Rd
A* A G C T T
Hpa I
Haemophilus parainflenzae
G T T * A A C
Kpn I
Klebsiella pneumoniae
G G T A C * C
Mbo I
Moraxella bovis
*G A T C
Pst I
Providencia stuartii
C T G C A * G
Sma I
Serratia marcescens
C C C * G G G
SstI
Streptomyces stanford
G A G C T * C
Sal I
Streptomyces albus G
G * T C G A C
Taq I
Thermophilus aquaticus
T * C G A
Xma I
Xanthamonas malvacearum
C * C C G G

25. Polymerase Chain Reaction ?

26. POLYMERASE CHAIN REACTION
STEPS INVOLVED
Reverse Primer
Forward Primer
Taq Polymerase

27. Unlike most enzymes, Taq polymerase can withstand high temperatures
necessary for DNA strand separation and can be left in the reaction.

28. PCR: Representative Temperature Profile

29. POLYMERASE CHAIN REACTION
AMPLIFICATION OF DNA

30. POLYMERASE CHAIN REACTION (Con’t)

31. EXPONENTIAL AMPLIFICATION OF PRODUCT

32. What reagents are missing?
STEPS INVOLVED
Taq Polymerase
Forward Primer
Reverse Primer
 template DNA
 primer 1
 primer 2
 dNTP
 Taq polymerase ?

33. Role of MgCl2 in PCR What could happen if [MgCl2] is too low
or too high?

34. DESIGNING PRIMERS
1.  Primers should be bases in length;   2.  Base composition should be 50-60% (G+C);   3.  Primers should end (3') in CG or GC: this creates “tight” ends and increases efficiency of priming   4.  Tm = 4(G + C) + 2(A + T) oC: Ta should be 2-5 oC below Tm( 55-80oC) 5.  Primers should not be complementary: primer-dimer
6.  Primer self-complementary (ability to form hairpins) should be avoided. 
Adapted from Innis and Gelfand, 1991

35. AGAROSE GEL ELECTROPHORESIS
Agarose: A polysaccharide extracted from seaweed.
Used to separate DNA fragments based on size

36. Detecting DNA using Ethidium Bromide
Structure of Ethidium Bromide
(Fluorescent Dye)
Intercalates between bases of DNA
When excited by UV light, EtBr emits fluorescent light at 590 nm

37. How are different sizes of DNA strands separated on agarose gel?
Mixture of DNA
molecules

38. DNA separation is based on fragment size
Decreasing Size
(+)
(-)
PCR1
PCR2

39. Varying concentrations of agarose
DNA Ladders
How does one make a 0.7%, 1.0% & 1.5%
agarose gel?
Why are there varying concentrations
of agarose gels?

40. Varying concentrations of agarose
DNA Ladders
How does one make a 0.7%, 1.0% & 1.5%
agarose gel?
Why are there varying concentrations
of agarose gels?
(Depends on length of fragment to be
separated)
Higher concentrations provide better
resolution for smaller DNA fragments
Lower concentrations provide better
resolution for larger DNA fragments

42. TIPS ON HOW TO DO WELL IN “WARM-UP” LAB

43. of microcentrifuge tube
The major reason for this experiment not to work is not having all the reaction components in the correct tube.
Place droplets on wall
of microcentrifuge tube
Tick list

44.  Component
 Reaction 1
 Reaction 2
 template DNA
 1ml
 primer 1
 primer 2  -
 dNTP
 2ml
 10X taq buffer
 5ml
 50mM MgCl2
 3ml
 water
 37.8ml
 36.8ml
 Taq DNA polymerase
 0.2ml
 Total volume
 50ml
Make Master Mix containing dNTPs, Buffer, MgCl2:
To save time, you can calculate how much of each to add
before coming to the lab.

45. Tick list for PCR Reactions
 Component
 Reaction 1
 Reaction 2
 template DNA
 1ml √
 1ml
 primer 1
 primer 2  -
 dNTP
 2ml
 10X taq buffer
 5ml
 50mM MgCl2
 3ml
 water
 37.8ml
 36.8ml
 Taq DNA polymerase
 0.2ml
 Total volume
 50ml

46. Tick list for DNA Digest
 Tube 1
Tube 2
Tube 3
 Tube 4
Tube 5
 DNA
 1ml √
 1ml
 10X buffer
 2ml √
2ml √
 2ml
 EcoRI
 0.5ml √
 0.5ml
 BamH1
 HindIII
 water
 16.5ml
 16ml
 total
 20ml

47. Droplet method Check pipet tip for solution.
Tap eppendorf
Check pipet tip for solution.
Eject solution on the side of tube.
Check for a droplet
To get the solution in the droplet to the bottom of the tube tap the tube on the bench.

48. Please Balance the Microcentrifuge

49. Please Balance the Microcentrifuge
WHY?
Non-Scientific Explanation
Scientific Explanation
The Microcentrifuge will break! ?

50. Please Balance the Microcentrifuge
WHY?
Non-Scientific Explanation
Scientific Explanation
Centripetal Force (CPF) exerted by sample
towards the centre of microcentrifuge.
Newtons 3rd law : Every action has
equal & opposite Rxn (For every CPF there
Should be equal & opposite CFF)
The Microcentrifuge
will break
Centripetal Force that the sample
exerts on the rotor depends on both
mass and radius.

51. THE END