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2004-2005AP Biology

Advanced Techniques

Electrophoresis, PCR,

Southern Blot, Sequencing,

RFLPs, Microarrays

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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

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2004-2005AP Biology

Gel Electrophoresis

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2004-2005AP Biology

Gel Electrophoresis

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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

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2004-2005AP Biology

RFLP

! Restriction Fragment Length Polymorphism

" change in DNA sequence affects

restriction enzyme “cut” site

" will create different band pattern

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2004-2005AP Biology

Southern Blot

! Want to locate a sequence on a gel?

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2004-2005AP Biology

Southern blot

! Transfer DNA from gel tofilter paper

! hybridize filter paper withtagged probe

" fragment with matchingsequence ‘lights up”

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2004-2005AP Biology

Polymerase Chain Reaction (PCR)

! What if you havetoo little DNA towork with?

" PCR is a methodfor making manycopies of aspecificsegment of DNA

" ~only need 1 cellof DNA to start

copying DNA without

bacteria or plasmids!

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2004-2005AP Biology

PCR primers

play DNAi movie

! The primers are critical!

" need to know sequence tomake proper primers

" primers flank targetsequence! 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/cycle

30 sec/step

Taq = Thermus aquaticus (an Archaebactera)

Highly thermostable – withstands temperatures up to 95°C for more

than 40min.

BTW, Taq is patented by Roche and is very expensive. Its usually the

largest consumable expense in a genomics lab. I’ve heard stories of

contraband Taq clones, so scientists could grow up their own

bacteria to produce Taq in the lab. It’s like pirated software -- pirated

genes!

PCR is an incredibly versatile technique:

An important use of PCR now is to “pull out” a piece of DNA

sequence, like a gene, from a larger collection of DNA, like the whole

cellular genome. You don’t have to go through the process of

restriction digest anymore to cut the gene out of the cellular DNA. You

can just define the gene with “flanking” primers and get a lot of

copies 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.

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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 solved

a core problem in genetics: How to make copies of a strand of DNA

you are interested in.

The existing methods were slow, expensive & imprecise. PCR turns

the job over to the very biomolecules that nature uses for copying

DNA: two "primers" that flag the beginning & end of the DNA stretch to

be copied; DNA polymerase that walks along the segment of DNA,

reading its code & assembling a copy; and a pile of DNA building

blocks 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, the

PCR can generate 100 billion similar molecules in an afternoon. The

reaction is easy to execute. It requires no more than a test tube, a few

simple reagents and a source of heat. The DNA sample that one

wishes to copy can be pure, or it can be a minute part of an extremely

complex mixture of biological materials. The DNA may come from a

hospital tissue specimen, from a single human hair, from a drop of

dried blood at the scene of a crime, from the tissues of a mummified

brain or from a 40,000-year-old wooly mammoth frozen in a glacier."

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2004-2005AP Biology

DNA Sequencing

! Sanger method

" determine the

nucleotide

sequence of DNA

" synthesize

comple mentary

strand of DNA in

vitro

" use tagged

bases

! ddNTP

dideoxynucleotides

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2004-2005AP Biology

Dideoxynucleotides

play Sequencing movie

! reagent mix for DNA replication

" “normal” N-bases

" dideoxy N-bases

! missing O for bonding of next

nucleotide

! terminates chain

! radioactively tagged

" DNA polymerase

" primer

" buffers & salt

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2004-2005AP Biology

Reading the sequence! Load gel with sequences from

ddA, ddT, ddC, ddG in separatelanes

" read lanes manually & carefully

" polyacrylamide gel

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2004-2005AP Biology

Fred Sanger

This was his 2nd Nobel Prize!!

" 1st was in 1958 for the

protein structure of insulin

1978|1980

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2004-2005AP Biology

Advancements to sequencing! Fluorescent tagging

" no more radioactivity

" all 4 bases in 1 lane! each base a different color

! Automated reading

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2004-2005AP Biology

Applied Biosystems, Inc

(ABI) built an industry on

these machines

Advancements to sequencing! Capillary tube electrophoresis

" no more pouring gels

" higher capacity & faster

384 lanes

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2004-2005AP Biology

PUBLIC

! Joint Genome Institute(DOE)

!MIT

!Washington Universityof St. Louis

!Baylor College ofMedicine

!Sanger Center (UK)

PRIVATE

!Celera Genomics

! Big labs!

" economy of scale

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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 toolsto 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

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2004-2005AP Biology

Different approaches“shot gun”“map-based”

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2004-2005AP Biology

Human Genome Project

On June 26, 2001, HGP published the “working

draft” of the DNA sequence of the human

genome.

Historic Event!

" blueprint

of a human

" the potential to

change science &

medicine

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2004-2005AP Biology

GenBank

Database of

genetic

sequences

gathered

from

research

Publicly

available!

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2004-2005AP Biology

The Progress

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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 genomes

complete

122+ bacterial

genomes

Data from NCBI and TIGR

(www.ncbi.nlm.nih.gov and www.tigr.org )

first eukaryote complete

(yeast)

first metazoan complete

(flatworm)

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eukaryotic

genomes

complete

or near

completion

including

Homo

sapiens,

mouse and

fruit 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 has

grown exponentially, including the growth of

online databases and resources. In this slide

we have the growth of the total number of base

pairs and the total number of genomes

completed since the beginning of the Human

Genome Project. As you can see, the sheer

number and growth of this resource has been

impressive—and daunting—in the last 5 years.

Few scientists are aware of, or make full use

of, all the open-source and public resources

available to them through the internet. The

Annual Nucleic Acids Research Database

issue listing contained 548 databases this

year!!

And, as this quote mentions, only half of those

who use the databases are familiar with their

tools. This Wellcome Trust study also made it

clear that many people become users of a

database after being told about it by

colleagues.

“Despite the large amount of publicity

surrounding the

Human Genome Project, a recent survey

conducted on behalf

of the Wellcome Trust indicates that only

half of biomedical

researchers using genome databases are

familiar with the

tools that can be used to actually access

the data.

In “The Molecular Biology Database

Collection: 2003 update”

by Andreas D. Baxevanis in the Jan 1, 2003

NAR database issue.

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2004-2005AP Biology

Other genomes

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2004-2005AP Biology

Microarrays

! Measuring

expression of genes

in 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 1980s

Realized quickly he needed an automated system: robot

spotter

Designed spotter & put plans on Internet for benefit of

scientific community.

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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

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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.

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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!

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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, cloned

the gene for Human Insulin. Genentech licensed the human insulin

technology to Eli Lilly, where it was named "Humulin" or

Recombinant Human Insulin. In 1982, human insulin became the

first recombinant DNA drug approved by FDA. Today, Humulin is

made in Indianapolis in gigantic fermentation vats, 4 stories high and

filled with bacteria!!! These fermentation vats operate 24 hours a day,

year round. The human insulin protein made by the E. coli bacteria is

collected from the vats, purified, and packaged for use by patients

with diabetes.

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2004-2005AP Biology

What next?

! After you have cloned & amplified DNA(genes), you can then tackle moreinteresting questions

" how does gene differ from person toperson?! …or species to species

" is a certain allele associated with ahereditary disorder

" in which cells is gene expressed?

" where is gene in genome?