Chapter 6

AP Biology 2005-2006

Chapter 6

Biotechnology:DNA Technology & Genomics

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The BIG Questions… How can we use our knowledge of DNA to:

diagnose disease or defect? cure disease or defect? change/improve organisms?

What are the techniques & applications of biotechnology?

Biotechnology The use of living organisms (or substances

from living organisms) for practical purposes

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Biotechnology Use of organisms to create a

desired product is not new humans have been doing

this for thousands of years Alcohol fermentation (for

brewing beer and food preservation)

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Biotechnology Agriculture

Plant and animal breeding Selective breeding – altered the genomes of

crops by breeding them with other plants.

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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 chapter is a survey of those tools…

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Bioengineering Tool kit Basic Tools

restriction enzymes ligase plasmids / cloning DNA databases / probes

Advanced Tools PCR DNA sequencing gel electrophoresis Southern blotting microarrays

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Isolating DNABefore DNA can be manipulated, it needs to be

isolated from cells.

1. 1. Cell membranes are disrupted use a detergent Salt used to neutralize lipids

2. 2. DNA precipitation ethanol used to dehydrate and aggregate DNA Salt used to neutralize phosphate groups in DNA

3. DNA isolation / storage

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DNA Isolation


1. Cut DNA To study the functions

of individual genes, molecular biologists will cut desired genes out of

a genomeplace the gene into

bacterial plasmidproduce recombinant

DNA


1. Cut DNA treat isolated DNA

with restriction enzymes restriction

endonucleases Evolved in bacteria

Immune System protection against

viruses & other bacteria


Restriction enzymes - Action cut DNA at specific sequences

Restriction (recognition) site Are usually 4-8 bp in length

produces protruding ends sticky ends that contain DNA

nucleotides that lack complementary bases

Some do not produce protruding ends blunt ends

CTGAATTCCGGACTTAAGGC

CTG|AATTCCGGACTTAA|GGC

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Restriction Enzymes

Many different enzymes named after

organism they are found in EcoRI,

HindIII, BamHI, SmaI

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Restriction Enzyme Cutting

sticky ends – enzyme digests (cuts) to make overhangs

EcoRI 5’ G A A T T C 3’3’ C T T A A G 5’

5’ G 3’ 5’ A A T T C 3’

3’ C T T A A 5’ 3’ G 5’

5’ overhang

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Restriction Enzyme Cutting

PstI 5’ C T G C A G 3’3’ G A C G T C 5’

5’ C T G C A 3’ 5’ G 3’3’ G 5’ 3’ A C G T C 5’

3’ overhang


2. Paste DNA Sticky ends allow:

H bonds between complementary bases to anneal

DNA Ligase enzyme “seals”

strands bonds sugar-

phosphate backbone together

Condensation reaction

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DNA Ligase T4 DNA ligase

originated in T4 bacteriophages

used to chemically join two blunt ends of DNA together


AATTC

AATTC

AATTC

GAATTC

G

G

GG

G

GAATTC

CTTAAG

GAATTCCTTAAG

CTTAA

CTTAA

CTTAAG

DNA ligasejoins the strands.

DNA

Sticky ends (complementarysingle-stranded DNA tails)

Recombinant DNA molecule

AATTCGG

CTTAA

Biotech use of restriction enzymes

Restriction enzymecuts the DNA

Add DNA from another source cut with same restriction enzyme

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Exercise 1

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Cut, Paste, Copy, Find… Word processing metaphor…

1. cut Isolate desired DNA restriction enzymes

2. paste ligase

3. copy plasmids

bacteria transformation

PCR 4. find

Southern blotting / probes 19

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Remember… Prokaryotic genomes (e.g. bacteria)

contain Chromosome Plasmids

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Plasmid

Chromosome

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Plasmid pBR322

Plasmids Contain “accessory

genes” Antibiotic resistance

Can carry and express foreign genes Plasmids are vectors

Vehicles by which DNA can be introduced into host cells

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Plasmid Restriction Maps

Shows the location of cleavage sites for many different enzymes These maps are used

like road maps to the DNA molecule

Numbers beside restriction enzyme indicates at which base pair the DNA is cut by that particular enzyme

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Sample Problem: Determine the size and

number of fragments that would be produced if the plasmid was digested with: EcoRV and Pvu II?

2 cuts, therefore 2 fragments

Between sites:

2064-185=1879 bp Rest:

4361-1879=2482 bp

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Why bacteria? Plasmid uptake is easy Cheap Reproduce rapidly and

frequently Produce multiple

copies of the recombinant DNA and protein in a short amount of time Recombinant

DNA and proteins are kept in large storage database to be used later

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Bacterium

Bacterialchromosome

Plasmid

Cell containing geneof interest

RecombinantDNA (plasmid)

Gene of interest

DNA ofchromosome

Recombinatebacterium

Protein harvested

Basic research on protein

Gene of interest

Copies of gene

Basic research on gene

Gene for pestresistance inserted

into plants

Gene used to alterbacteria for cleaning

up toxic waste

Protein dissolvesblood clots in heart

attack therapy

Human growth hormone treatsstunted growth

Protein expressedby gene of interest

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Protein Databases

Actin Protein

Unnamed Protein

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Review: Transformation vs. Recombination

Transformation the introduction of foreign DNA (usually

a plasmid) into a bacterial cell

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Recombination Fragment of DNA

composed of sequences originating from at least two different sources

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3. Copying (Cloning)

1 Isolate plasmid DNA and human DNA.

2 Cut both DNA samples with the same restriction enzyme

3 Mix the DNAs; they join by base pairing. The products are recombinant plasmids andmany nonrecombinant plasmids.

TECHNIQUE In this example, a human gene is inserted into a plasmid from E. coli. The plasmid contains the ampR gene, which makes E. coli cells resistant to the antibiotic ampicillin. It also contains the lacZ gene, which encodes -galactosidase. This enzyme hydrolyzes a molecular mimic of lactose (X-gal) to form a blue product. Only three plasmids and three human DNA fragments are shown, but millions of copies of the plasmid and a mixture of millions of different human DNA fragments would be present in the samples.

Stickyends Human DNA

fragments

Human cell

Gene of interest

Bacterial cell

ampR gene(ampicillinresistance)

Bacterial plasmid

Restriction site

Recombinant DNA plasmids

lacZ gene (lactose breakdown)

Figure 20.4

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RESULTS Only a cell that took up a plasmid, which has the ampR gene, will reproduce and form a colony. Colonies with nonrecombinant plasmids will be blue, because they can hydrolyze X-gal. Colonies with recombinant plasmids, in which lacZ is disrupted, will be white, because they cannot hydrolyze X-gal. By screening the white colonies with a nucleic acid probe (see Figure 20.5), researchers can identify clones of bacterial cells carrying the gene of interest.

Colony carrying non-recombinant plasmid with intact lacZ gene

Bacterialclone

Colony carryingrecombinant plasmidwith disrupted lacZ gene

Recombinantbacteria

4Introduce the DNA into bacterial cells (transoformation).

5 Plate the bacteria on agar containingampicillin and X-gal (lactose). Incubate untilcolonies grow.

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How do we know if it worked? We know cloning worked if:

Transformation has occurred i.e. there has been plasmid

uptake into the bacteria Recombination has

occurred i.e. foreign genes have been

inserted into the bacterial plasmid

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Selecting for successful transformation

Antibiotic resistance genes as a selectable marker

Plasmid has both “added” gene & antibiotic resistance gene

selection

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Amp SelectionIf bacteria don’t pick up

plasmidthey will not have

antibiotic resistancedie on antibiotic (amp)

platesIf bacteria pick up

plasmid survive on antibiotic

(amp) plates

We have selected only those colonies that have undergone successful transformation

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Amp Selection

Ampicillin is the selecting agent

LB/amp plateLB plate

all bacteria grow

only transformed bacteria grow

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Screening for successful recombination

Transformed colonies have both

recombinant and non-recombinant plasmids

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recombinant non-recombinant


LacZ Screening

LacZ gene codes for β galactosidase

Lactose (or X-gal) is our screening agent

plasmid

ampresistance

LacZ gene

restriction sites

lactose blue color

recombinantplasmid

ampresistance

brokenLacZ gene

lactose white colorX

insertedgeneof interest

origin ofreplication

all in LacZ geneEcoRIBamHI

HindIII

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LacZ screening system

XX Make sure inserted plasmid is

recombinant plasmid LacZ gene on plasmid

produces digestive enzyme lactose(X-gal) blue blue colonies

insert foreign DNA into LacZ gene breaks gene lactose (X-gal) blue white colonies

white bacterial colonies have recombinant plasmid

We want

these

!!

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Amp selection & LacZ screening

- gene of interest

- LacZ gene

- amp resistance

LB/amp LB/amp/Xgal

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Cut, Paste, Copy, Find… Word processing metaphor…

cut restriction enzymes

paste ligase

copy plasmids

bacteria transformation

PCR find

Southern blotting / probes

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4. Find White colonies

could have recombinant plasmids desired and

undesired genes

How do you find the conony with the gene of interest?

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4. Find

recombinant plasmids inserted into bacteria

gene of interest

bacterial colonies (clones) grown on LB/amp/Xgal petri plates

But howdo we find

colony with our gene of interest

in it?

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DNA hybridization find gene in bacterial colony using a probe

short, single stranded DNA molecule complementary to part of gene of interest tagged with radioactive P32 or fluorescence

heat treat genomic DNA unwinds (denatures) strands

DNA hybridization between probe & denatured DNA

Locating your gene of interest

5’3’

labeled probe

genomic DNAG A T C A G T A G

C T A G T C A T C


HybridizationCloning- plate with bacterial

colonies carrying recombinant plasmids

1

2

Hybridization- heat filter paper to

denature DNA- wash filter paper with

radioactive probe which will only attach to gene of interest

Replicate plate- press filter paper onto

plate to take sample of cells from every colony

3

Locate- expose film- locate colony on plate

from film

4

film

filter

plate

plate + filter

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Classwork/Homework Pg. 281 #1-3 Pg. 282 #6,7,10 Pg. 287 #16,18 Pg. 289 #20,21 Pg. 291 #2-7,10,11(b,c),15,16(a,b),17,19 Pg. 295 #4,5

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Chapter 6