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Page 1: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

Module  1:  DNA  Engineering  M1D1  Lecture  

Experiments  and  lectures  based  upon  research  in  Prof.  Bevin  Engelward’s  laboratory  

   

Some  lecture  slides  from    Prof.  Engelward  and  Samson  

Page 2: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

•  Lectures  in  Module  1  – Biology  and  engineering  of  DNA  damage  and  repair  – Preview  of  experiments  each  day  

•  Today:  M1D1  Lecture  – Preview  of  module  and  underlying  biology  

•  Today/Tomorrow:  M1D1  Lab    – PCR  basics  – RaIonal  primer  design  

Page 3: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

What experimental question will you ask in Module 1?

What is the frequency of DNA repair by homologous recombination in mouse embryonic stem cells?

This raises the following questions

•  How does DNA get damaged?

•  What is DNA repair?

•  Why does DNA repair exist?

•  Why do we care about how efficient DNA repair is?

•  How does one actually measure DNA repair?

Julian  Voss-­‐Andreae  (photographer,  sculptor)  

Page 4: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

Key Experimental Methods for Module 1

•  Construction of truncated eGFP gene – Cloning

•  PCR, Restriction Enzymes, Ligation/Transformation, Bacterial culture

•  Mammalian tissue cell culture

•  Transfecting plasmids into mammalian cells

•  Flow cytometry to measure DNA repair

•  Statistical analysis of biological data

Page 5: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

DNA  structure  –  stable,  but  vulnerable  

Page 6: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

DNA  structure  –  stable,  but  vulnerable  

Page 7: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

Sunlight

Pollution & Food

Inflammation

Oxidative Radicals

Cigarette Smoke

Radiation

Courtesy  of  Bevin  Engelward  

DNA  is  constantly  being  damaged  by  endogenous  and  exogenous  agents  

Page 8: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

What’s  the  big  deal?  

Page 9: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

The  main  DNA  repair  pathways:  

Page 10: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

Homologous Recombination

Page 11: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

DNA  Damage  and  Repair  

Direct-Reversal repair (DR)

Single-strand break Alkylation

Adapted from Hoeijmakers 2001 Nature

Recombina*onal  (HR,  NHEJ)  

Page 12: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

DNA  Damage  and  Repair  

Direct-Reversal repair (DR)

Single-strand break Alkylation

Adapted from Hoeijmakers 2001 Nature

Recombina*onal  (HR,  NHEJ)  

Page 13: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

DNA  Damage  and  Repair  

Direct-Reversal repair (DR)

Single-strand break Alkylation

Adapted from Hoeijmakers 2001 Nature

Recombina*onal  (HR,  NHEJ)  

Page 14: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

DNA  Damage  and  Repair  

Direct-Reversal repair (DR)

Single-strand break Alkylation

Adapted from Hoeijmakers 2001 Nature

Recombina*onal  (HR,  NHEJ)  

Page 15: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

What  are  the  known  risk  factors  for  Skin  Cancer?  

Page 16: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

What  are  the  known  risk  factors  for  Skin  Cancer?  

*Joke  shamelessly  borrowed  from  Leona  Samson  

Page 17: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’
Page 18: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

Errol  C.  Friedberg  Nature  Reviews  Cancer  1,  22-­‐33  (2001)  

NucleoIde  Excision  Repair  

 XPA  XPB  XPC  XPD  XPE  XPF  XPG  

Page 19: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

Lack  of  DNA  repair  accelerates  the  onset  of  cancer  

Xeroderma  pigmentosum  (XP)  

Errol  C.  Friedberg  Nature  Reviews  Cancer  1,  22-­‐33  (2001)  

Page 20: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

XP frequency = ~1:250,000 giving a theoretical maximum of how many cases worldwide with 2,000-fold increased risk

Even if just 1% of the population is relatively repair deficient, could have how many with several-fold increased risk

Adapted  from  GROSSMAN  and  Wei  (1995)  Clinical  Chem  41:  1854-­‐1863  

Page 21: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

XP frequency = ~1:250,000 giving a theoretical maximum of ~28,000 cases worldwide with 2,000-fold increased risk

Even if just 1% of the population is relatively repair deficient, could have tens of millions with several-fold increased risk

Adapted  from  GROSSMAN  and  Wei  (1995)  Clinical  Chem  41:  1854-­‐1863  

Page 22: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

Reactivation of UV damaged DNA by Host cell Reactivation (HCR)

+ UV light Transient

transfection peripheral

blood lymphocytes

Time to repair CAT Assay

Athas & GROSSMAN Cancer Res. 1991

Page 23: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

Cells from XP patients

Cells from “healthy” people

Athas & GROSSMAN

Page 24: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

Cells from XP patients

Athas & GROSSMAN

RelaIvely  HIGH  repair  

RelaIvely  LOW  repair  

Page 25: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

XP frequency = ~1:250,000 giving a theoretical maximum of ~28,000 cases worldwide with 2,000-fold increased risk

Even if just 1% of the population is relatively repair deficient, could have tens of millions with several-fold increased risk

Adapted  from  GROSSMAN  and  Wei  (1995)  Clinical  Chem  41:  1854-­‐1863  

Page 26: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

Wei  Q,  Matanoski  GM,  Farmer  ER,  HedayaI  MA,  GROSSMAN  L.  Proc  Natl  Acad  Sci  U  S  A.  1993  90:1614-­‐8.    

Low NER status combined with excessive sun exposure is very dangerous

**P<0.001  

Page 27: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

…and the effect is gender specific.

Wei  Q,  Matanoski  GM,  Farmer  ER,  HedayaI  MA,  GROSSMAN  L.  Proc  Natl  Acad  Sci  U  S  A.  1993  90:1614-­‐8.    

Page 28: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

Very current motivation for Mod1: NYT  –  Monday,  Sept  8  2014  –  Winners  of  the  Lasker  Prize  (“American  Nobel”)  announced  

Dr.  King  discovered  link  between  BRCA1  and  breast  cancer.  

MutaIons  in  BRCA1  &  BRCA2  increase  risk  of  breast  cancer  –  but  mutaIon  does  not  guarantee  cancer.      Is  there  a  beker  way  to  decide  on  preempIve  acIon  other  than  just  geneIc  screening?  

Page 29: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

Your Experiment:

Create a plasmid that will be part of a homologous recombination sensor.

Measure the frequency of cells in which HR between two plasmids gives rise to a fluorescent cell.

Test conditions that might affect the frequency of green cells!

Page 30: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

Your Experiment:

Page 31: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

Today you will start building your system.

Page 32: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

M1D1: PCR

DNA  Learning  Center  -­‐-­‐  Cold  Spring  Harbor  

Page 33: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

hkp://www.sparknotes.com/biology/molecular/dnareplicaIonandrepair/secIon2.rhtml  

DNA  ReplicaIon  

Page 34: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

M1D1: Primer Design

Page 35: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

M1D1: Primer Design

Page 36: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

M1D1: Primer Design

Page 37: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

M1D1: Primer Design

Page 38: Module’1:’DNA’Engineering’ M1D1’Lecture’...• Lectures’in’Module’1’ – Biology’and’engineering’of’DNA’damage’and’repair’ – Preview’of’experiments’each’day’

Image  credits:    Slide  2:            Slide  32:    

"Steel  Jellyfish",  2006.  Stainless  steel,  height:  4'7"  (1.40  m).  LocaIon:  Friday  Harbor  Labs,  San  Juan  Island,  WA  hkp://commons.wikimedia.org/wiki/File:Steel_Jellyfish_%28GFP%29.jpg  

hkp://chemwiki.ucdavis.edu/Organic_Chemistry/Organic_Chemistry_With_a_Biological_Emphasis/Chapter_10%3A_Phosphoryl_transfer_reacIons/SecIon_10.4%3A_Phosphate_diesters  


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