DNA Technology and Genetic Engineering

porcupineideaBiotechnology

Dec 16, 2012 (4 years and 9 months ago)

135 views

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Use 2: To identify people

Example: Forensic analysis (DNA
fingerprinting)

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2) Steps in DNA Identification


A) Isolate DNA and make copies


B) Cut the DNA into shorter fragments that
contain
RFLPs


C) Sort the DNA by size


D) Compare samples to identify a person

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A) Why Make Copies of DNA?


Sometimes samples of DNA are too scarce to use
for identification


Drop of blood


Skin cells, etc


Scientists can use the polymerase chain reaction
(PCR)


Creates billions of copies of a particular DNA
fragment


A fragment


NOT the whole genome



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Steps of PCR


A) Denature (separate strands)


NOT with
helicase


With 95
°
C heat!


B) Anneal (prime strands)


Primers serve as starting point
for DNA polymerase


With custom* DNA primers at 50
-
65
°
C


C) Elongate with DNA
polymerase at 72
°
C


Add free nucleotides to tube


Does DNA polymerase denature?

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PCR: Exponential Amplification!


PCR mixture put in
thermocycler


Denature, anneal, elongate


repeat 20
-
35x


How many DNA molecules?


Original DNA sample


2 fragments after 1
round


2
nd

round: 2 fragments


4


3
rd
: 8


35
th
: 34 billion... from 1 original molecule!

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Steps of PCR


The
polymerase
chain reaction
(PCR) can
quickly clone a
small sample
of DNA in a
test tube

Initial

DNA

segment

1

2

4

8

Number of DNA molecules

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2) Cut DNA into Fragments with
known
RFLPs


RFLP = restriction fragment length
polymorphism


Human genome: 98% non
-
coding DNA


Length between genes can vary due to short
stretches of DNA sequence that can repeat a
few or many times


Can PCR known regions that contain these
repeats


Select restriction enzymes that cleave
around them, creating
RFLPs


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RFLPs

and Restriction Enzymes

Allele 1

Allele 2

w

x

y

Cut

Cut

Cut

z

y

DNA from chromosomes

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How Can We See Our DNA pieces?


Gel electrophoresis
-

separates fragments
by size using electricity


DNA samples dyed and loaded into wells in
a jello
-
like gel (agarose)


DNA fragments move through gel


Bigger pieces can’t move through gel very
quickly


stay near top


Smaller pieces move faster and are near the
bottom of the gel

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Review: What is DNA made of?

DNA nucleotides:

1)
Deoxyribose sugar

2)
Nitrogenous base

3)
Phosphate

2

1

3

Does DNA have a charge?

YES!

NEGATIVELY CHARGED

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Gel electrophoresis

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Gel electrophoresis


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1

2

Longer

fragments

Shorter

fragments

Allele 1

Allele 2

w

x

y

Cut

Cut

Cut

z

y

DNA from
chromosomes

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FBI probes 13 regions in the genome



Approximate probability that two people will
match at a random DNA sample in one site is 1/10



The probability that two people will match at
three sites is: 1/10 x 1/10 x 1/10 = 1/1000



Applying this probability to all 13 sites is one in
ten trillion (or virtually 0)!

DNA

Fingerprinting

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DNA Fingerprint

1. Victim

2. Suspect 1

3. Blood on suspect 1

4. Blood on suspect 2

5. Suspect 2


1

2

3

4

5

Which suspect

is guilty?

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Is the defendant innocent or guilty?

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What if there are too many DNA
pieces?


What if there are so many fragments you
can’t see individual ones?


Can label specific fragments with a
radioactive probe


After gel is run, you can transfer the DNA to
a nylon membrane and “probe” for the
RFLP sequences

Digest with restriction enzyme X

A

B

C

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Paternity tests


Who is the father?

C/AF = Child and Alleged
Father DNA mixed and
run in same well

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Use 3: To identify human
genetic diseases

Screening for sickle
-
cell anemia

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G
AATTC

Wild
-
type allele

Mutant allele

CTTAA
C

Restriction site

Not a restriction site

A single nucleotide change can
make a difference


GA
C
TTC

CT
G
AAC





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Example: Sickle
-
cell allele destroys
an
Mst
II site


Southern blot

Gel electrophoresis

Which are the

right bands?

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RFLP mapping of sickle
-
cell
anemia in a family

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Use 4: To find and identify all
human genes

The Human Genome Project

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Human genome facts


Genome Pioneer = Eric Lander (MIT!)


Started in 1990 and projected to take 15
years to finish. Working draft in 2000 and
declared complete in 2003.


Goal to sequence all of our DNA, which is
over 3 billion nucleotides!


Originally we thought we had over 100,000
genes, but we actually have ~20,000 genes

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Human genome facts


A decade later, what do we know?


Known single
-
gene diseases jumped from 100
-
3000!


More than 200 genes have been linked to cancer
(3x what was known before)


Many diseases linked to extra or missing copies of
genes


Genetic basis for disease can lead to targeted
drug therapies (long way to go though)

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Use 5: Change characteristics

Transgenic organisms


These transgenic
sheep carry a gene
for a human blood
protein, which they
secrete in their milk.
This protein inhibits
an enzyme that
contributes to lung
damage in patients
with cystic fibrosis,
emphysema, and
other chronic
respiratory diseases.

Pharming

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Golden rice

Transgenic rice
produces beta
carotene (we use it
to make vitamin A)
that gives the rice
their golden color
and their increased
nutritional value.
400 million people
suffer from Vitamin
A deficiency leads
to blindness and
infection.

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To insert normal

genes into mutant
cells



Gene Therapy

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Risks and Ethical Concerns


Genetic engineering involves some risks


Risk to the researcher


use crippled
strains


Possible ecological damage from pollen
transfer between GM and wild crops


superweeds


Pollen from a transgenic variety of corn
that contains a pesticide may stunt or kill
monarch caterpillars


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Risks and Ethical Concerns


Human genome project also raises some
issues


How can we be sure that a transferred
gene makes the appropriate amount of
protein and is expressed at the right
time/place?


What do we reserve gene therapy for:
serious diseases only, athletic
enhancement, appearance, intelligence?


Reduced genetic diversity?


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Summary of the applications of
DNA technology


Medicine
-

bacteria produce human
proteins


To identify people


To diagnose genetic diseases


To find and identify all of the human genes


Gene therapy


Genetically engineered foods


Agriculture


bacteria that decompose
nitrogen faster, disease and pest resistant
plants, bigger, sweeter, more nutritious
plants, bigger, leaner animals


Industry


bacteria used to break down
pollutants