Download Linkage map - Cloudfront.net

Genetic Technology
Manipulating Genes
A. Genetic Engineering

Genetic engineering (AKA recombinant
DNA technology) is faster & more reliable
method of selecting certain trait in
population
• Artificial selection is done by humans breeding
specific individuals with certain traits - slow
• Natural selection is nature selecting specific
individuals with certain traits – very slow
•
Genetic engineering involves cleaving (cutting
out) DNA from one organism into small
fragments & inserting desired gene into host
DNA of same or different species
Desired gene
Host DNA


Also called recombinant DNA technology
since DNA gets recombined to make one new
one
If plants or animals contain foreign DNA from
this technology, are called transgenic
organisms or genetically modified organism
(GMO)
• Example is tobacco plant that contains
glowing gene from firefly – plant glows!!
Example of Transgenic
Organisms
Zebra Fish
Firefly
Bioluminescence
Tobacco Plant
Caterpillar
B. Steps of Engineering

Making transgenic or GMO takes 3 steps:
1. cleave DNA – isolate DNA fragment
2. vector - attach DNA fragment to carrier
3. insertion - insert DNA into host organism
3. Insert into host
2. Make a vector
1. Cleave DNA
1.
DNA Cleavage
 Must isolate small parts of DNA (DNA can
contain millions of base pairs
•
use special enzymes called restriction
enzymes that cut both sides of dsDNA at
specific areas of nucleotide sequence
•
depending on which way DNA is cut, get 2
different ends:
o
sticky end
o
blunt end
o
sticky end
 dsDNA
is cut leaving some single strands
 can only do that if there is a palindrome = letter
order written same way backwards as forwards


ex: “mom” backwards is “mom” but with dsDNA, both
sides are included
“GAATTC” on top side (forwards) and “CTTAAG” on
bottom side (backwards)
A T C C A G G A A T T C C A A G C T C
T A G G T C C T T A A G G T T C G A G

A
T
restriction enzyme recognizes specific palindromes and
will cut somewhere within there
 ex: EcoRI recognizes GAATTC & will cut in b/t G – A
on both sides leaving sticky ends ready to bond
T C C A G G A A T T C C A
A G G T C C T T A A G G T
A
T
G C
C G
T
A
C
G
After Cleaving
A
T
A A T T C C A A G C T C
T C C A G G
G G T T C G A G
A G G T C C T T A A

TTAA and AATT sticky end have nothing to
bond to, so if same restriction enzyme cuts
DNA of organism and host’s organism, both
sticky ends will match so bonding will be
easier
Example of Sticky ends
o
blunt end

A
T
DNA is cut all way through like with
scissors
T C C A G G A C T T C C A
A G G T C C T G A A G G T
A
T
G
C
C
G
T
A
C
G
After Cleaving
A
T
T C C A G G A
A G G T C C T

C T T C C A A G C T C
G A A G G T T C G A G
both ends are bonded with other bases so are blunt
Examples of Restriction
Enzymes
2. Attach to vector


Loose fragments of DNA
need to be attached to
vector (carrier) first
Two types of vectors:
o
o
Biological vector: bacteria
plasmid or virus
Mechanical vector:
micropipette or microscopic
metal bullet

Since both DNA and vector were cleaved
with same restriction enzyme, both ends will
match
•
Join pieces using DNA ligase
3.
Insertion into host
 Recombined plasmid (or other vector) is
inserted into host’s cell
 When host replicates, inserted DNA also
replicates producing more of that desired
gene
• bacterial plasmid can replicate every 20
min!
o
Bacterial plasmid
 Inserts
plasmid into
bacteria’s cytoplasm
o
Virus
 Injects
DNA directly
into host’s DNA
 Process called
transduction

Plasmid can replicate 500 times per cell, and
each clone replicates 500 times… and so on
• Clone: genetically identical copies of
original
Dolly (1996-2003)
The first ever
cloned animal

can also replicate DNA segments by
using Polymerase Chain Reaction
(PCR)
• dsDNA strands are separated
(unzipped) by heat
• special heat-resistant enzymes
replicate DNA
• important advancement technique
used to match DNA with very little
DNA to begin with
 Don’t need much DNA from crime
scenes
PCR Technique
Example of Recombinant DNA
C. Uses for Genetic Engineering

recombinant DNA (genetic engineering) is
currently useful in many areas of life
1.
2.
3.
Industry
Medicine
Agriculture
1. Industry
a.
b.
c.
d.
clothing: bacteria E. coli are transgenic
with DNA to make indigo dye
 indigo dye in nature is VERY
expensive, so can make blue jeans
cheaply
food: making corn that has high protein
content (corn is mostly carbohydrate)
fuel: use corn husks to make fuel for
cars
sewage: clean water using bacteria
2. Medicine
a.
b.
c.
d.
hormone: can produce human growth
hormone (hGH) to treat people with growth
disorders (Achondroplasia, Turner
syndrome)
medicine: produce human insulin (formerly
bovine/cow) with bacterial plasmids
diseases: transgenic sheep are produced
that produce Factor VIII protein for
hemophiliacs
Vaccines: remove virus’ dangerous genes
3. Agriculture





making more/bigger/healthier/fresher food
Crops resistant to viruses and insects
canola plants make more canola oil
peanuts & soybeans that don’t cause
allergic reactions
corn that can grow with very little water
(survive drought)
D. The Human Genome Project

International effort started in1990,
Human Genome Project (HGP)
was organized to completely map
and sequence human genome
• complete sequence of nucleotides
(3.2 billion) in human DNA
(completed 2000).
• Complete map of 20,000 genes
(2006) on 23 sets of chromosomes
Human Genome Project
Leaders of the Genome
Project (Dr. Landers
and Dr. Collins
How did they do that?



1. How did they find out that genes U-Z are on
chromosome set #2 and not on set #8?
2. How do we know gene N is next to M and O and
not somewhere else?
A
B
C
D
A
B
C
D
U
V
W
X
Y
Z
U
V
W
X
Y
Z
Q
R
S
Q
R
S
Set# 2
ANSWER: Linkage Maps
Set# 8
L
M
N
O
P
L
M
N
O
P
Linkage Map

Linkage map: genetic map that shows relative
locations of genes on a chromosome
• Found locations of genes on specific
chromosomes, but didn’t know the order
o
o
•
Gene M is on chromosome 11
Where on chromosome 11 is gene M?
Can find RELATIVE order of genes from a
linkage map
Linkage Map
Chromosome
11
Chromosome
M

Using PCR, can make millions of copies of
DNA fragments to find patterns in certain
genes
o Use genetic markers to trace inheritance of
genes, which shows us where that gene is
located relative to the others
• Father has genes M
& HD
• Mother does not
 Out of 5 kids, 3
inherited M,
and of those 3,
2 also got HD
 M & HD are
close to each
other
Gel Electrophoresis

Process of separating DNA fragments to compare sizes and
therefore similarities
• Electricity is sent through gel containing DNA fragments
• DNA pieces will migrate toward bottom
o Smaller pieces will “run” faster
o Larger pieces will be stuck toward top
large
small
Genetic Markers
Paternity Tests
Results: D2 not Dad’s
Results: S2 adopted
How did they sequence it?

sequencing human genome compares DNA
fragments to each other
• pieces that overlap are pieced together
ABCD
NOPQ
DEFGHIJ
ABCDEFGHIJKLMNOPQRSTUVWXYZ
IJKLMNOP
RSTUVWX
WXYZ
BCD
LMNOPQRS
All these
fragments
came from
cleaving
DNA into
little
workable
pieces
By lining up
pieces that
overlap, can
get entire
sequence
E. Applications of HGP

Diagnosing genetic disorders – individuals find out if
they are carrying gene for specific disease
 Can be done for fetuses using epithelial cells (from
amniotic fluid)
 Dilemma – do YOU want to know if you have gene
for cancer or heart disease?
 Pros: can alter lifestyle NOW to help prevent
cancer or heart disease from coming
 Cons: always in fear about what may or may
not happen

Gene therapy – inserting normal genes into
human cells to correct genetic disorder
 Cystic
fibrosis, sickle-cell anemia, hemophilia,
AIDS, cancer, heart disease are all being
studied as genetic diseases in which gene
therapy may work
Gene Therapy

DNA fingerprinting – compare unknown DNA to known
DNA to find out if they match
 DNA cut by restriction enzymes would show same sizes
each time (same palindrome sequence)
 Called restriction fragment length polymorphisms
(RFLPs)
o Solve crimes
o Maternity/paternity
DNA Fingerprinting
Paternity Tests
Results: D2 not Dad’s
Results: S2 adopted