1

2004-2005AP Biology

Advanced Techniques

Electrophoresis, PCR,Southern Blot, Sequencing,

RFLPs, Microarrays

2

2004-2005AP Biology

Gel Electrophoresis Separation of DNA fragments by size

DNA is negatively charged moves toward + charge in electrical field

agarose gel “swimming through Jello” smaller fragments move faster

cut DNA with restriction enzymes

3

2004-2005AP Biology

Gel Electrophoresis

4

2004-2005AP Biology

Gel Electrophoresis

5

2004-2005AP Biology

Measuring fragment size compare bands to a known “standard”

usually lambda phage cut with HindIII nice range of sizes with a distinct pattern

6

2004-2005AP Biology

RFLP Restriction Fragment Length Polymorphism

change in DNA sequence affectsrestriction enzyme “cut” site

will create different band pattern

7

2004-2005AP Biology

Southern Blot Want to locate a sequence on a gel?

8

2004-2005AP Biology

Southern blot Transfer DNA from gel to

filter paper hybridize filter paper with

tagged probe fragment with matching

sequence ‘lights up”

9

2004-2005AP Biology

Polymerase Chain Reaction (PCR) What if you have

too little DNA towork with? PCR is a method

for making manycopies of aspecificsegment of DNA

~only need 1 cellof DNA to start

copying DNA withoutbacteria or plasmids!

10

2004-2005AP Biology

PCR primers

play DNAi movie

The primers are critical! need to know sequence to

make proper primers primers flank target

sequence start with long piece of DNA &

copy a specified shortersegment

primers define section of DNAto be cloned

Taq polymerase from hot springs bacteria why do we use it? 20-30 cycles

3 steps/cycle30 sec/step

Taq = Thermus aquaticus (an Archaebactera)Highly thermostable – withstands temperatures up to 95°C for morethan 40min.BTW, Taq is patented by Roche and is very expensive. Its usually thelargest consumable expense in a genomics lab. I’ve heard stories ofcontraband Taq clones, so scientists could grow up their ownbacteria to produce Taq in the lab. It’s like pirated software -- piratedgenes!

PCR is an incredibly versatile technique:An important use of PCR now is to “pull out” a piece of DNAsequence, like a gene, from a larger collection of DNA, like the wholecellular genome. You don’t have to go through the process ofrestriction digest anymore to cut the gene out of the cellular DNA. Youcan just define the gene with “flanking” primers and get a lot ofcopies in 40 minutes through PCR.

Note: You can also add in a restriction site to the copies of the gene(if one doesn’t exist) by adding them at the end of the original primers.

11

2004-2005AP Biology

Kary Mullis development of PCR technique

a copying machine for DNA

1985|1993

In 1985, Kary Mullis invented a process he called PCR, which solveda core problem in genetics: How to make copies of a strand of DNAyou are interested in.The existing methods were slow, expensive & imprecise. PCR turnsthe job over to the very biomolecules that nature uses for copyingDNA: two "primers" that flag the beginning & end of the DNA stretch tobe copied; DNA polymerase that walks along the segment of DNA,reading its code & assembling a copy; and a pile of DNA buildingblocks that the polymerase needs to make that copy.As he wrote later in Scientific American:"Beginning with a single molecule of the genetic material DNA, thePCR can generate 100 billion similar molecules in an afternoon. Thereaction is easy to execute. It requires no more than a test tube, a fewsimple reagents and a source of heat. The DNA sample that onewishes to copy can be pure, or it can be a minute part of an extremelycomplex mixture of biological materials. The DNA may come from ahospital tissue specimen, from a single human hair, from a drop ofdried blood at the scene of a crime, from the tissues of a mummifiedbrain or from a 40,000-year-old wooly mammoth frozen in a glacier."

12

2004-2005AP Biology

DNA Sequencing Sanger method

determine thenucleotidesequence of DNA

synthesizecomple mentarystrand of DNA invitro

use taggedbases ddNTP

dideoxynucleotides

13

2004-2005AP Biology

Dideoxynucleotides

play Sequencing movie

reagent mix for DNA replication “normal” N-bases dideoxy N-bases

missing O for bonding of nextnucleotide

terminates chain radioactively tagged

DNA polymerase primer buffers & salt

14

2004-2005AP Biology

Reading the sequence Load gel with sequences from

ddA, ddT, ddC, ddG in separatelanes read lanes manually & carefully polyacrylamide gel

15

2004-2005AP Biology

Fred SangerThis was his 2nd Nobel Prize!!

1st was in 1958 for theprotein structure of insulin

1978|1980

16

2004-2005AP Biology

Advancements to sequencing Fluorescent tagging

no more radioactivity all 4 bases in 1 lane

each base a different color Automated reading

17

2004-2005AP Biology

Applied Biosystems, Inc(ABI) built an industry onthese machines

Advancements to sequencing Capillary tube electrophoresis

no more pouring gels higher capacity & faster

384 lanes

18

2004-2005AP Biology

PUBLIC Joint Genome Institute

(DOE)MITWashington University

of St. LouisBaylor College of

MedicineSanger Center (UK)PRIVATECelera Genomics

Big labs! economy of scale

19

2004-2005AP Biology

Human Genome Project U.S government project

begun in 1990 estimated to be a 15 year project

DOE & NIH initiated by Jim Watson led by Francis Collins

goal was to sequence entirehuman genome 3 billion base pairs

Celera Genomics Craig Venter challenged gov’t would do it faster, cheaper private company

1990-1995 build the technology groundwork

improve sequencing methods build clones build better data management systems (computer tools

to find overlaps) better, cheaper, faster!

1996-1998 painstaking sequencing work

1998 Celera genomics challenge

2000 rough draft of human genome (90% sequence, 99% accurate)

2001 1st draft of human genome

2003 “finished” sequence of human genome can’t sequence telomeres & centromeres

20

2004-2005AP Biology

Different approaches“shot gun”“map-based”

21

2004-2005AP Biology

Human Genome ProjectOn June 26, 2001, HGP published the “workingdraft” of the DNA sequence of the humangenome.

Historic Event! blueprint

of a human the potential to

change science &medicine

22

2004-2005AP Biology

GenBankDatabase ofgeneticsequencesgatheredfromresearch

Publiclyavailable!

23

2004-2005AP Biology

The Progress

Dec-82

Dec-83

Dec-84

Dec-85

Dec-86

Dec-87

Dec-88

Dec-89

Dec-90

Dec-91

Dec-92

Dec-93

Dec-94

Dec-95

Dec-96

Dec-97

Dec-98

Dec-99

Dec-00

Dec-01

Dec-02

Jun-03

S1

0.E+00

5.E+09

1.E+10

2.E+10

2.E+10

3.E+10

3.E+10

4.E+10

First 2 bacterial genomescomplete

122+ bacterialgenomes

Data from NCBI and TIGR(www.ncbi.nlm.nih.gov and www.tigr.org )

first eukaryote complete(yeast)

first metazoan complete(flatworm)

17eukaryoticgenomescompleteor nearcompletionincludingHomosapiens,mouse andfruit fly

Official “15 year”Human Genome Project:

1990-2003.

# of DNA base pairs (billions)

in GenBank

In the last 5 years or so the amount of data hasgrown exponentially, including the growth ofonline databases and resources. In this slidewe have the growth of the total number of basepairs and the total number of genomescompleted since the beginning of the HumanGenome Project. As you can see, the sheernumber and growth of this resource has beenimpressive—and daunting—in the last 5 years.

Few scientists are aware of, or make full useof, all the open-source and public resourcesavailable to them through the internet. TheAnnual Nucleic Acids Research Databaseissue listing contained 548 databases thisyear!!

And, as this quote mentions, only half of thosewho use the databases are familiar with theirtools. This Wellcome Trust study also made itclear that many people become users of adatabase after being told about it bycolleagues.

“Despite the large amount of publicitysurrounding theHuman Genome Project, a recent surveyconducted on behalfof the Wellcome Trust indicates that onlyhalf of biomedicalresearchers using genome databases arefamiliar with thetools that can be used to actually accessthe data.

In “The Molecular Biology DatabaseCollection: 2003 update”by Andreas D. Baxevanis in the Jan 1, 2003NAR database issue.

24

2004-2005AP Biology

Other genomes

25

2004-2005AP Biology

Microarrays Measuring

expression of genesin a tissue sample samples of mRNA

from cells make cDNA 2-color fluorescent

tagging hybridize to spots

of genes colored spots =

gene expression red, green, yellow

Developed by Pat Brown at Stanford in late 1980sRealized quickly he needed an automated system: robotspotterDesigned spotter & put plans on Internet for benefit ofscientific community.

26

2004-2005AP Biology

When to use microarrays? Research tool

it’s all about comparisons gene expression….

before vs. after treatment cancer vs. normal cells wound healing vs. scarring stages of development

color coding red = expression in 1 sample green = expression in other sample yellow = expression in both

samples dark = low expression in both

27

2004-2005AP Biology

DNA Chip Patented microarray technology from

Affymetrix automated DNA synthesis of genes of

interest on chip chips are more consistent smaller spots/more spots

per chip can buy specific chips

human chip mouse chip etc.

28

2004-2005AP Biology

Biotechnology today: Applications Application of DNA technologies

basic biological research medical diagnostics medical treatment (gene therapy) pharmaceutical production forensics environmental cleanup agricultural applications

…and then there’s the ethics issues!

29

2004-2005AP Biology

Application of recombinant DNA Combining sequences of DNA from

2 different sources into 1 DNA molecule often from different species

human insulin gene in E. coli (humulin) frost resistant gene from Arctic fish in

strawberries “Roundup-ready” bacterial gene in soybeans BT bacterial gene in corn jellyfish glow gene in

Zebra “Glofish”

In 1978, scientists at the Biotechnology Company, Genentech, clonedthe gene for Human Insulin. Genentech licensed the human insulintechnology to Eli Lilly, where it was named "Humulin" orRecombinant Human Insulin. In 1982, human insulin became thefirst recombinant DNA drug approved by FDA. Today, Humulin ismade in Indianapolis in gigantic fermentation vats, 4 stories high andfilled with bacteria!!! These fermentation vats operate 24 hours a day,year round. The human insulin protein made by the E. coli bacteria iscollected from the vats, purified, and packaged for use by patientswith diabetes.

30

2004-2005AP Biology

What next? After you have cloned & amplified DNA

(genes), you can then tackle moreinteresting questions how does gene differ from person to

person? …or species to species

is a certain allele associated with ahereditary disorder

in which cells is gene expressed? where is gene in genome?