Biotechnology Gel Electrophoresis

lowlytoolboxBiotechnology

Oct 22, 2013 (4 years and 2 months ago)

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AP Biology

2007
-
2008

Biotechnology


Gel Electrophoresis

How We Analyze DNA So That We Can Do Stuff
With It.

AP Biology

A brave new world?

AP Biology

Biotechnology


Manipulating genes to suit our purposes is
nothing new.


Crossbreeding / Selective Breeding


Picking organisms with traits you like and breeding
them


Goal is to get babies with traits that you REALLY
like.


Trouble is that Cross/Selective breeding is not
terribly precise


You may not get the results you want


May recombine other traits in ways that were not
intended, etc.


AP Biology

Biotechnology today


Genetic Engineering


Direct manipulation of DNA


Allows much more specific control over
particular genes


Fewer unintended results


However, if you are going to engineer DNA &
genes & organisms, then you need a

set of tools

to work with


this is a look at one

of those tools…

Our tool kit…

AP Biology

To manipulate DNA, we first need to be
able to cut it up


WHY?


Really it’s our only means of being able to
analyze it


Also, if we’re going to take certain parts that
we like OUT of one organism’s DNA and stick
them into another organism’s DNA, then we
need to be able to “cut and paste”


So how the heck can we cut up DNA??


Breaking out the scissors will NOT work.


The answer is….


ENZYMES!

AP Biology

How do we cut DNA?


Restriction enzymes


restriction endonucleases


discovered in 1960s


Occur naturally in bacteria


Protect bacteria against viruses

& other bacteria

AP Biology

So what do restriction enzymes do?


Do they just hack DNA up into bits?


Well, sort of, but they only do it at
certain, very predictable, locations
along the DNA sequence


The places where the restriction
enzymes cut DNA are called restriction
sites


Specific sequences of “letters” on the DNA
strand recognized by Restriction enzymes

AP Biology

Restriction enzymes


Action of enzyme


cut DNA at specific sequences


restriction site


Many different kinds of Restriction
enzymes


named after organism


in which they are found

Madam I’m Adam

CT
GAATTC
CG

GA
CTTAAG
GC

CT
G
|
AATTC
CG

GA
CTTAA
|
G
GC





AP Biology

Many uses of restriction enzymes…


Now that we can cut DNA with restriction
enzymes…


Not only can we cut DNA out of the genome of
one organism and stick it in another…


Glow in the dark jellyfish gene inside of a mouse


Human insulin gene inside of bacteria


But we can also cut up DNA from different
people… or different organisms…

and
compare it


For what purpose?


forensics


medical diagnostics


paternity


evolutionary relationships


and more…

AP Biology

How do we compare cut up DNA?


Compare DNA fragments by separating them
according to SIZE.


So, how do we separate DNA fragments
according to size?


We can’t see all the little


pieces and pick them out


with our fingers!


run them through a “strainer”


to sort them


Gelatin is the strainer


gel electrophoresis

AP Biology

Gel electrophoresis


A method of separating DNA
in a gelatin
-
like material
using an electrical field


DNA is negatively charged


when it’s in an electrical
field it moves toward the
positive side

+



DNA
















“swimming through Jello”

AP Biology


DNA moves in an electrical field…


so how does that help you compare DNA
fragments?


size of DNA fragment affects how far it travels


small pieces travel more quickly through the gel


large pieces travel slower & lag behind

Gel electrophoresis

+



DNA














“swimming through Jello”

AP Biology

Gel Electrophoresis

longer fragments

shorter fragments

power

source

completed gel

gel

DNA &

restriction enzyme

wells

-

+

AP Biology

Gene 1

GCTTGTAAC
GGCC
T
CATCATCAT
TCGCC
GGCC
TACGCTT

CGAACATTG
CCGG
A
GTAGTAGTA
AGCGG
CCGG
ATGCGA

repeats

Different people have different DNA

cut sites

cut sites

GCTTGTAAC
G GCC
T
CATCATCAT
CGCC
G GCC
TACGCTT

CGAACATTG
CCG G
A
GTAGTAGTA
GCGG
CCG G
ATGCGA

1

2

3

DNA





+

gene 1

Cut the DNA

Great…But how does the
fact that different size
pieces of DNA move at
different speeds through a
gel HELP us ANALYZE
the DNA????

AP Biology

Gene 1

GCTTGTAAC
GGCC
T
CATCATCAT
TCGCC
GGCC
TACGCTT

CGAACATTG
CCGG
A
GTAGTAGTA
AGCGG
CCGG
ATGCGA

Differences between people

cut sites

cut sites

DNA





+

allele 1

Gene 2

GCTTGTAAC
GGCC
T
CATCATCATCATCATCAT
CC
GGCC
T

CGAACATTG
CCGG
A
GTAGTAGTAGTAGTAGTA
GG
CCGG

DNA fingerprint

allele 2

1

2

3

AP Biology

Uses: Evolutionary relationships


Comparing DNA samples from different
organisms to measure evolutionary
relationships



+

DNA



1

3

2

4

5

1

2

3

4

5

turtle

snake

rat

squirrel

fruitfly

AP Biology

Uses: Medical diagnostic (genetic testing)


Comparing normal allele to disease allele

chromosome with

disease
-
causing

gene 2

chromosome

with normal

gene 1



+

DNA



Example: genetic test for Huntington’s disease

AP Biology

Uses: Forensics


Comparing DNA sample from crime
scene with suspects & victim



+

S1

DNA



S2

S3

V

suspects

crime

scene

sample

AP Biology

DNA fingerprints


Comparing blood
samples on
defendant’s clothing
to determine if it
belongs to victim


DNA fingerprinting


comparing DNA
banding pattern
between different
individuals


~unique patterns

AP Biology

Electrophoresis use in forensics


Evidence from murder trial


Do you think suspect is guilty?

“standard”

blood sample 3 from crime scene

“standard”

blood sample 1 from crime scene

blood sample 2 from crime scene

blood sample from victim 2

blood sample from victim 1

blood sample from suspect

OJ Simpson

N Brown

R Goldman

AP Biology

Uses: Paternity


Who’s the father?

+

DNA



child

Mom

F1

F2



AP Biology

2007
-
2008

I’m a
-
glow
!

Got any Questions?

AP Biology

In reality, much of each person’s DNA
is identical to every other person…


Don’t want the genes for important proteins to be messed up, do
we?????


So How/Why is each person’s DNA pattern different?


There are big sections of “junk” DNA within our genomes


“Junk” DNA doesn’t code for proteins


made up of repeated patterns


CAT, GCC, and others


each person may have different number of repeats


Why are these repeated patterns there? Not entirely clear…


many sites are found on our 23 chromosome pairs with

different repeat patterns

GCTTGTAACGGCCT
CATCATCAT
TCGCCGGCCTACGCTT

CGAACATTGCCGGA
GTAGTAGTA
AGCGGCCGGATGCGA

GCTTGTAACGG
CATCATCATCATCATCAT
CCGGCCTACG

CGAACATTGCC
GTAGTAGTAGTAGTAGTA
GGCCGGATGC