CCHS AP Biology Goldberg

Chapter XXBiotechnology:

Genomics & DNA Technology

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 entire human genome 3 billion base pairs

Celera Genomics

Craig Venter challenged gov’t

would do it faster, cheaper

private company

Different Approaches

3. Assemble DNA sequence using overlapping sequences.

“map-based method”gov’t method

“shotgun method”Craig Venter’s method

1. Cut DNA from entire chromosome

into small fragments and clone.

2. Sequence each segment & arrange

based on overlapping nucleotide

sequences.

1. Cut chromosomal DNA segment into

fragments, arrange based on

overlapping nucleotide sequences,

and clone fragments.

2. Cut and clone into smaller fragments.

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

GenBank

Database of

genetic

sequences

gathered

from

research

Publicly

available!

Organizing the Data

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And we didn’t stop with us! How does our genome stack up?

Organism

Genome Size

(bases)

Estimated

Genes

Human (Homo sapiens) 3 billion ~20,000

Laboratory mouse (M. musculus) 2.6 billion ~20,000

Mustard weed (A. thaliana) 100 million ~25,000

Roundworm (C. elegans) 97 million 19,000

Fruit fly (D. melanogaster) 137 million 13,000

Yeast (S. cerevisiae) 12.1 million 6,000

Bacterium (E. coli) 4.6 million 3,200

Human Immunodeficiency Virus (HIV) 9700 9

Interspersed Repetitive DNA

Repetitive DNA is spread throughout genome

interspersed repetitive DNA (SINEs Short INterspersed Elements) make up 25-40% of mammalian genome

in humans, at least 5% of genome is made of a family of similar sequences called, Alu elements (PV92 anyone?!) 300 bases long

Alu is an example of a "jumping gene" called a transposon; a DNA sequence that "reproduces" by copying itself & inserting into new chromosome locations

Rearrangements in the Genome

Transposons

transposable genetic element

piece of DNA that can move from one

location to another in cell’s genome

One gene of an insertion sequence codes for transposase, which catalyzes the

transposon’s movement. The inverted repeats, about 20 to 40 nucleotide pairs long,

are backward, upside-down versions of each other. In transposition, transposase

molecules bind to the inverted repeats & catalyze the cutting & resealing of DNA

required for insertion of the transposon at a target site.

Transposons

Insertion of

transposon

sequence in new

position in genome

Insertion sequences

cause mutations

when they happen to

land within the

coding sequence of a

gene or within a DNA

region that regulates

gene expression.

Transposons

Barbara McClintock

discovered 1st transposons in Zea mays

(corn) in 1947

1947 | 1983

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Families of Genes

Human globin gene family

evolved from duplication of common ancestral globin gene

Different versions are

expressed at different

times in development

allowing hemoglobin to

function throughout life

of developing animal

The BIG Questions…

How can we use our knowledge of DNA to:

diagnose disease or genetic defect?

cure disease or genetic defect?

change/improve organisms?

What are the techniques & applications of

biotechnology?

direct manipulation of genes for practical

purposes

Biotechnology

Genetic manipulation of organisms is not new

humans have been doing this for thousands of years plant & animal breeding

Evolution & Breeding of Food Plants

Evolution of Zea mays from ancestral teosinte (left) to

modern corn (right). The middle figure shows possible

hybrids of teosinte & early corn varieties

artificial selection!

Evolution & Breeding of Food Plants

“Descendants” of the wild mustard

Brassica genus

artificial selection!

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Animal Husbandry / Breeding

artificial selection!

Biotechnology Today

Genetic Engineering

direct manipulation of DNA

if you are going to engineer DNA &

genes & organisms, then you need a

set of tools to work with

this unit is a survey

of those tools…

Our tool kit…

Bioengineering Tool Kit

Basic Tools

restriction enzymes

ligase

gel electrophoresis

plasmids for gene cloning

Advanced Tools

PCR

DNA sequencing

Southern blotting

DNA libraries / probes

microarrays

Cut, Paste, Copy, Find…

Word processing metaphor…

cut (Ctrl + X) restriction enzymes

paste (Ctrl + V) ligase

copy (Ctrl + C) via PCR

via plasmids bacteria

transformation

find (Ctrl + F) Southern blotting

probes

Cutting DNA

Restriction enzymes

restriction endonucleases

discovered in 1960s

evolved in bacteria to cut up foreign DNA (“action restricted to foreign DNA”)

protection against viruses & other bacteriabacteria protect their

own DNA by methylation& by not using the base sequences recognized by the enzymes in their own DNA

Paste DNA

Sticky ends allow:

H bonds between complementary bases to anneal

Ligase

enzyme “seals” strands bonds sugar-

phosphate bonds

covalent bond of DNA backbone

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AATTC

AATTC

AATTC

GAATTC

G

G

G

G

G

GAATTC

CTTAAG

GAATTC

CTTAAG

CTTAA

CTTAA

CTTAAG

DNA ligasejoins the strands.

DNA

Sticky ends (complementarysingle-stranded DNA tails)

Recombinant DNA molecule

Biotech Use of Restriction Enzymes

Restriction enzymecuts the DNA

Add DNA from another source cut with same

restriction enzyme

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” – GFP!

Development of GFP

Shimomura, Chalfie, Tsien

discovery, isolation, and purification of

GFP and many fluorescent analogs

1961, 1994 | 2008

Osamu Shimomura Martin Chalfie Roger Tsien

Cut, Paste, Copy, Find…

Word processing metaphor…

cut restriction enzymes

paste ligase

copy PCR

plasmids bacteria

transformation

find Southern blotting

cDNA probes

Plasmids

Plasmids

small supplemental circles of DNA

5000 - 20,000 base pairs

self-replicating

carry extra genes

2-30 genes

can be exchanged between bacteria

bacterial ‘sex’!!

rapid evolution

antibiotic resistance

can be imported

from environment

Biotechnology

Used to insert new genes into

bacteria

example: pUC18

engineered plasmid used in biotech

antibiotic

resistance gene on

plasmid is used as

a selective agent

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Transformation

Bacteria are opportunists

pick up naked foreign DNA wherever it

may be hanging out

some have surface transport proteins that

are specialized for the uptake of naked DNA

import bits of chromosomes from other

bacteria

incorporate the DNA bits into their own

chromosome

express new gene

form of recombination

Swapping DNA

Genetic recombination by trading DNA

1 3 2

arg+

trp-

arg-

trp+

minimal

media

Copy DNA

Plasmids

small, self-replicating

circular DNA molecules

insert DNA sequence into plasmid

vector = “vehicle” into organism

transformation

insert recombinant plasmid into bacteria

bacteria make lots of copies of plasmid

grow recombinant bacteria on agar plate

clone of cells = lots of bacteria

production of many copies of inserted gene

DNA RNA protein trait

Recombinant PlasmidAntibiotic resistance genes as a selectable marker

Restriction sites for splicing in gene of interest

Selectable marker Plasmid has both

“added” gene &

antibiotic resistance

gene

If bacteria don’t pick

up plasmid then “die”

on antibiotic plates

If bacteria pick up

plasmid then survive on

antibiotic plates

selecting for successful

transformation

selection

GFP

Selection for Plasmid Uptake

Ampicillin becomes a selecting agent

only bacteria with the plasmid will grow

on amp plate

LB/amp plateLB plate

all bacteria grow

only transformed

bacteria grow

Gene Cloning