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Last year’s headline @ CNN (9/20/2010). In a heartbeat 15.59 % (2,167 votes) If the FDA approves it, I guess it's okay 10.45 % (1,453 votes) I'll wait a while and see how people fare 21.34 % (2,966 votes) In a pinch 4.99 % (693 votes) Not on your life 47.63 % (6,621 votes).

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Last year s headline @ cnn 9 20 2010 l.jpg
Last year’s headline @ CNN (9/20/2010)

  • In a heartbeat 15.59% (2,167 votes)

  • If the FDA approves it, I guess it's okay 10.45% (1,453 votes)

  • I'll wait a while and see how people fare 21.34% (2,966 votes)

  • In a pinch 4.99% (693 votes)

  • Not on your life 47.63% (6,621 votes)

Clarified: What does "genetically modified" salmon mean?

Public opinion poll: Would you eat genetically modified salmon?

http://eatocracy.cnn.com/2010/09/20/genetically-modified-salmon/?hpt=T2

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Chapter 2 technical foundations of genomics l.jpg

Chapter 2Technical Foundations of Genomics

Recombinant-DNA techniques used in genomics

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Contents l.jpg
Contents

  • Introduction

  • Genomic and cDNA libraries

  • DNA Hybridization and Northern blots

  • Subcloning in vectors

  • Restriction-enzyme mapping

  • DNA sequencing

  • PCR amplification

  • Protein expression

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


What is the main goal of genomics l.jpg
What is the main goal of genomics?

  • Sequence the entire genome by cutting it into small, manageable pieces (fragments)

  • Assemble the entire genome from the pieces (fragments)

  • Make sense of the genome

  • Understand how gene expression takes place?

  • How life processes are networked?

  • Understand life??

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Introduction l.jpg
Introduction

  • Genomics built on recombinant-DNA technology (developed since early 1970s)

  • Thorough understanding of recombinant-DNA techniques

    • Prerequisite for understanding genomics technologies

  • Differences between genomics and recombinant-DNA technology

    • Genomics is high throughput approaches to allow more analyses in parallel

    • Genomics is dependent on computational analysis due to larger data sets

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Genomic and cdna libraries l.jpg
Genomic and cDNA libraries

  • Libraries are fragments of DNA cloned into a vector (microbial) but these are not organized according their natural arrangement on the chromosomes

  • Libraries are usually constructed before sequencing (prerequisite)

  • Genomic libraries are used for genomewide sequencing

  • cDNA libraries are needed for EST (expressed sequence tags) sequencing

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Central dogma l.jpg
Central Dogma

Vector

Genomic library

DNA

RNA

Proteins

Transcription

Vector

cDNA library

cDNA

mRNA

Translation

Expressed Sequence Tags

ESTs

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Genomic library l.jpg
Genomic library

  • Made from fragments of genomic DNA

    • Genomic DNA cut up with restriction enzymes or randomly broken by mechanical shearing (passing through syringe needle, or by sonication)

  • Fragments ligated into cloning vectors

    • Small insert

      • Lambda phage: 20-50 Kbp

      • Plasmid: ~10 Kbp

    • Large insert

      • BACs (Bactetial Artificial Chromosomes) 100-300 kbp

      • YACs (Yeast Artificial Chromosomes) ~ 1 MBP

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Restriction enzyme mapping l.jpg
Restriction-enzyme mapping

  • Used for physical mapping of DNA

  • Restriction enzymes (RE) cut at defined sites

    • Palindromic sequences

  • Sites are landmarks on DNA

  • Then fragments are separated by gel electrophoresis

CGATCG GTAC

GCTAGC CATG

Sticky end Versus blunt

3300 REs known but ~300 used

4 bp- 8 bp cutters

GCGCGCGCGC

CGCGCGCGCG

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


How to make a genomic library l.jpg
How to make a genomic library

ori

total genomic DNA

ampR

genomic DNA

restriction

enzyme

ori

anneal

and ligate

ampR

ori

plasmid (black)

ori

ori

ampR

ampR

ampR

same

restriction

enzyme

transform E. coli;

select for

Amp resistance

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Making a cdna library l.jpg
Making a cDNA library

  • Step 1: Isolate RNA

  • RNA is purified from tissue or cell line

  • The mRNA is then isolated away from ribosomal and tRNAs

  • Column with oligo dT is used to bind poly A

tissue or cell

mRNA

polyA

stationary support

polyT

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Step 2 obtain cdna from rna l.jpg
Step 2: Obtain cDNA from RNA

  • mRNA is treated with the enzyme reverse transcriptase (RT)

  • The enzyme copies sequence of mRNA into first strand of DNA

  • Digest RNA with RnaseH

  • Another enzyme (RT) is used to make second strand of cDNA

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


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Step 3: Transformation

  • Double-stranded cDNA is inserted into cloning vector

  • cDNA is ligated into cloning vector (plasmid or phage)

  • Vector is transformed or infected into bacteria

plasmid

E. Coli

bacteria

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Step 4 library screening both genomic and cdna l.jpg
Step 4: Library screening (both genomic and cDNA)

A=T

C=G

selected

colonies

  • Colony DNA is attached to membrane

  • DNA is screened with labeled probes

  • DNA is labeled with radioactivity

  • Labeled DNA is allowed to hybridize with DNA on membrane

  • After washing, positive hybridization spots are identified

membrane

Radioactive

probe

hybridization

X-ray film

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Cdna to est l.jpg
cDNA to EST

cDNA

library

  • For use in EST sequencing

    • Need to array individual clones

  • Library is spread on bacterial plates

  • Individual colonies are picked

  • Colonies are placed in test tubes or microtiter plates

Clone 1

2

3

4

5

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Colony picking l.jpg
Colony picking

  • Automatic colony pickers play key role in genomics

  • Instead of manually picking one colony at a time, they identify and pick multiple colonies from plates

  • Pickers then deposit each colony into a microtiter well

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Nucleic acid hybridization l.jpg
Nucleic Acid Hybridization

  • Basis of microarrays for determining gene expression

  • Process by which complementary strands find each other

  • A–T and C–G base pairing

  • speed and fidelity: dependent on temperature, salt, sequence, and concentration (High temp and low salt)

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Steps in northern blotting l.jpg
Steps in Northern blotting

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Northern blot l.jpg
Northern blot

  • Gene expression analyzed by Northern blots

  • RNA samples undergo electrophoresis

  • RNA separated by molecular weight

  • Transferred to membrane

  • Probe labeled

    • Radioactivity or antibody ligand

  • Hybridized to RNA on membrane

  • Hybridization speed and fidelity dependent on time, temperature, salt concentration, and nucleic acid sequence and concentration

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Northern blot example l.jpg
Northern blot example

Time after elicitation

  • Example of time course of gene induction

  • Upper panel: RNA after electrophoresis (18S and 28S rRNA)

  • Bands correspond to ribosomal RNA (EtBR)

  • Probe detects two bands

  • Lower panel: Lower band shows rapid induction and then decline

  • Upper band shows slower induction, but stays induced for longer

0 2 4 6 8 10 12 24

M

0 2 4 6 8 10 12 24

– 4.2 kb

– 2.1 kb

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Northern blot and microarray l.jpg
Northern blot and microarray

0 2 5 6 7 hrs

0 2 5 6 7 9 11 hrs

DMC1 –

SPS1 –

DIT1 –

SPS100 –

DMC1 –

SPS1 –

0 2 5 6 7 9 11 hrs

fold

repressed

fold

induced

DIT1 –

>20 10x 3x | 3x 10x >20

1:1

SPS100 –

Identify genes whose expression was induced during sporulation in yeast

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Cross hybridization l.jpg
Cross-hybridization

  • Hybridization to a related, but not identical, sequence = cross-hybridization

  • Example: A probe from one member of a gene family is likely to hybridize to all other members

  • Problem in microarrays, particularly cDNA arrays

  • Oligonucleotide arrays prescreened to eliminate sequences likely to cross-hybridize

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Subcloning l.jpg
Subcloning

  • Propagating fragments of cloned DNA

  • Used for sequencing and protein production

  • Plasmid vectors

    • Replicate in bacteria

    • Resistant to antibiotics

    • Cloning sites

ORI

Region

into which

DNA can

be inserted

Plasmid

cloning

vector

ampr

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Subcloning vector and fragment l.jpg
Subcloning: vector and fragment

restriction

enzymes

  • Vector and fragment to be inserted must have compatible ends

  • Sticky ends anneal

  • Enzyme ligase makes covalent bond between vector and fragment

  • Use of recombination instead of restriction sites

DNA

fragment

cloning

vector

recombinant

plasmid

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Recombination cloning l.jpg
Recombination cloning

  • Uses site-specific recombination for subcloning

  • DNA fragment flanked by recombination sites

  • Add recombinase “Clonase®”

  • Moves fragment from one vector to another

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Transformation into bacteria l.jpg
Transformation into bacteria

  • Bacteria prepared for transformation by making outer membrane permeable to DNA

    • Become competent

  • DNA added to bacteria

  • Heat shock (370 c)

  • Plate on selective media

E. coli

host cell

recombinant

plasmid

transformed cell

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Restriction enzyme mapping27 l.jpg
Restriction-enzyme mapping

  • Used for physical mapping of DNA

  • Restriction enzymes cut at defined sites

    • Palindromic sequences

  • Sites are landmarks on DNA

  • Then fragments are separated by gel electrophoresis

CGATCG

GCTAGC

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Gel electrophoresis l.jpg
Gel electrophoresis

  • DNA fragments are separated by size in electric field

  • DNA negatively charged: proportional to size of fragment

  • Separated through gel matrix

    • Agarose or acrylamide

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Cutting a bac with restriction enzymes l.jpg

.

.

Log MW

.

.

Distance

Cutting a BAC with restriction enzymes

  • Separate DNA fragments are cut with restriction enzyme

  • DNA is visualized with ethidium bromide

    • Binds to DNA and fluoresces orange

  • The sizes of the fragments are determined based on a standard

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Dna sequencing l.jpg
DNA sequencing

  • Most current sequencing projects use the chain termination method

    • Also known as Sanger sequencing, after its inventor, Fredrick Sanger

  • Based on action of DNA polymerase

    • Adds nucleotides to complementary strand

  • Requires template DNA and primer

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458



Slide32 l.jpg

Chain terminates Upper Saddle River, New Jersey 07458

H

dideoxyribonucleotide

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Chain termination l.jpg
Chain termination Upper Saddle River, New Jersey 07458

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Chain termination sequencing l.jpg
Chain-termination sequencing Upper Saddle River, New Jersey 07458

  • Dideoxynucleotides stop synthesis

    • Chain terminators

  • Included in amounts so as to terminate every time the base appears in the template

  • Use four reactions

    • One for each base: A,C,G, and T

Template

3’ ATCGGTGCATAGCTTGT 5’

5’ TAGCCACGTATCGAACA* 3’

5’ TAGCCACGTATCGAA* 3’

5’ TAGCCACGTATCGA* 3’

5’ TAGCCACGTA* 3’

5’ TAGCCA* 3’

5’ TA* 3’

Sequence reaction products

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Sequence separation l.jpg
Sequence separation Upper Saddle River, New Jersey 07458

  • Terminated chains need to be separated

  • Requires one-base-pair resolution

    • See difference between chains of X and X+1 base pairs

  • Gel electrophoresis

    • Very thin polyacryamide gel

    • High voltage

    • Works with radioactive or fluorescent labels

+

C A G T C A G T

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Sequence reading of radioactively labeled reactions l.jpg

A Upper Saddle River, New Jersey 07458

T

C

G

Sequence reading of radioactively labeled reactions

  • Radioactive labeled reactions

    • Gel dried

    • Placed on X-ray film

  • Sequence read from bottom up

  • Each lane is a different base

+

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Sequence detection l.jpg
Sequence detection Upper Saddle River, New Jersey 07458

  • To detect products of sequencing reaction

  • Include labeled nucleotides

  • Formerly, radioactive labels were used

  • Now fluorescent labels

  • Use different fluorescent tag for each nucleotide

  • Can run all four reactions in same lane

TAGCCACGTATCGAA*

TAGCCACGTATC*

TAGCCACG*

TAGCCACGT*

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Automated dna sequencing l.jpg
Automated DNA sequencing Upper Saddle River, New Jersey 07458

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Assume all these lanes are merged in one but different colored bands l.jpg

A Upper Saddle River, New Jersey 07458

T

C

G

Assume all these lanes are merged in one but different colored bands

A=blue

C= Green

G= yellow

T= red

+

Laser

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Sequence reading of fluorescently labeled reactions l.jpg
Sequence reading of fluorescently labeled reactions Upper Saddle River, New Jersey 07458

  • Fluorescently labeled reactions scanned by laser as particular point is passed

  • Color picked up by detector

  • Output sent directly to computer

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Summary of chain termination sequencing l.jpg
Summary of chain termination sequencing Upper Saddle River, New Jersey 07458

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Polymerase chain reaction l.jpg
Polymerase chain reaction Upper Saddle River, New Jersey 07458

  • Used in sequencing, diagnostics, comparative genomics, etc.

  • Uses thermostable DNA polymerase

    • Able to function near boiling temperature

  • Two primers complementary to sequences at 5’ and 3’ of region to be amplified

  • Double-stranded DNA template

  • Performed in thermal cyclers programmed to raise and lower temperature

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


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PCR machines Upper Saddle River, New Jersey 07458

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Pcr reaction annealing primers l.jpg
PCR reaction: annealing primers Upper Saddle River, New Jersey 07458

  • Template melted into two strands by high heat

    • > 90 degrees C

  • Primers anneal to both strands

  • Polymerase makes a copy of both strands

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Pcr reaction amplification l.jpg
PCR reaction: amplification Upper Saddle River, New Jersey 07458

  • Temperature raised to melt newly made DNA

  • Primers allowed to anneal as temperature drops

  • Polymerase elongates new second strand of DNA

  • Process repeated

  • Exponential increase in DNA

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458




Protein expression l.jpg
Protein expression Upper Saddle River, New Jersey 07458

  • Important for proteomics

  • Need large amounts of recombinant protein for the following:

    • Structure determination

    • Antibody production

    • Protein arrays

  • Proteins made in bacteria, yeast, and insect cells

  • Then must purify the recombinant protein away from other proteins

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Protein expression vectors l.jpg
Protein expression vectors Upper Saddle River, New Jersey 07458

  • Protein expression vectors have the following:

    • Inducible promoters

    • Tags for purification

      • Histidines

      • Epitopes: FLAG or myc

      • Proteins: Maltose binding proteins

    • Coding sequence inserted in frame

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Making recombinant protein l.jpg
Making recombinant protein Upper Saddle River, New Jersey 07458

  • Expression vector transformed into bacteria

  • Bacteria grown to saturation

  • Compound added for induction

    • e.g., IPTG

  • Protein accumulates in bacteria

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Protein purification l.jpg
Protein purification Upper Saddle River, New Jersey 07458

  • Contents of bacteria run over column

  • Tagged proteins bind to column

  • Examples

    • Nickel column for His-tagged proteins

    • Anti-myc antibody column for Myc-tagged proteins

  • Elution yields purified protein

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Recombinant protein l.jpg

SHR::MBP Upper Saddle River, New Jersey 07458

SDS-PAGE

1

2

4

5

6

7

kDa

124 –

83 –

42 –

Recombinant protein

  • Gel electrophoresis of recombinant protein shows the following:

    • Soluble proteins

    • Column flow-through

    • Purified protein

  • Four fractions from column

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


Summary l.jpg
Summary Upper Saddle River, New Jersey 07458

  • Libraries

  • Hybridization and Northern blots

  • Subcloning

  • Restriction-enzyme mapping

  • Sequencing

  • PCR

  • Protein expression

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458


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