AP Biology Chapter 20. Biotechnology: DNA Technology & Genomics.

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AP Biology Chapter 20. Biotechnology: DNA Technology & Genomics

Transcript of AP Biology Chapter 20. Biotechnology: DNA Technology & Genomics.

Page 1: AP Biology Chapter 20. Biotechnology: DNA Technology & Genomics.

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Chapter 20.

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?

direct manipulation of genes for practical purposes

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Biotechnology Genetic manipulation of organisms is

not new humans have been doing this for

thousands of years plant & animal breeding

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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

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Animal husbandry / breeding

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Biotechnology today Genetic Engineering

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…

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Bioengineering Tool kit Basic Tools

restriction enzymes ligase plasmids / cloning DNA libraries / probes

Advanced Tools PCR DNA sequencing gel electrophoresis Southern blotting microarrays

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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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Cut DNA Restriction enzymes

restriction endonucleases discovered in 1960s evolved in bacteria to cut up foreign

DNA (“restriction”) 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

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Restriction enzymes Action of enzyme

cut DNA at specific sequences restriction site

symmetrical “palindrome” produces protruding ends

sticky ends

Many different enzymes named after organism they are found in

EcoRI, HindIII, BamHI, SmaI

Madam I’m Adam

CTGAATTCCGGACTTAAGGC

CTG|AATTCCGGACTTAA|GGC

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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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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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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

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Gene cloningRecombinant DNA movie

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Human CloningHuman cloning is very controversial & not the main goal of biotechnology

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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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Advanced Techniques

Electrophoresis & RFLPs

Southern Blot, PCR,

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Gel Electrophoresis Separation of DNA fragments by size

DNA is negatively charged moves toward + charge in electrical field

agarose gel “swimming through Jello” smaller fragments move faster

cut DNA with restriction enzymes

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Gel Electrophoresis

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Gel Electrophoresis

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Measuring fragment size compare bands to a known “standard”

usually lambda phage virus cut with HindIII nice range of sizes with a distinct pattern

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RFLP Restriction Fragment Length Polymorphism

differences in DNA between individuals

change in DNA sequence affects restriction enzyme “cut” site

will create different band pattern

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Polymorphisms in populations Differences between individuals at the

DNA level

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RFLP use in forensics 1st case successfully using DNA evidence

1987 rape case convicting Tommie Lee Andrews

“standard”

“standard”

“standard”

“standard”

semen sample from rapist

semen sample from rapist

blood sample from suspect

blood sample from suspect

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RFLP use in forensics Evidence from murder trial

Do you think suspect is guilty?

“standard”

blood sample 3 from crime scene

blood sample from victim 2

blood sample from suspect

“standard”

blood sample 1 from crime scene

blood sample from victim 1

blood sample 2 from crime scene

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Southern Blot Want to locate a sequence on a gel?

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Southern blot Transfer DNA from gel to

filter paper hybridize filter paper with

tagged probe fragment with matching

sequence “lights up”

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Hybridization in Southern Blotting Use radioactive probe to locate gene on

filter paper go back to gel

& cut out piece of DNA you want to collect

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Polymerase Chain Reaction (PCR) What if you have

too little DNA to work with? PCR is a method

for making many copies of a specific segment of DNA

~only need 1 cell of DNA to start

copying DNA without bacteria or plasmids!

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PCR process It’s copying DNA in a test tube! What do you need?

template strand DNA polymerase enzyme nucleotides primer

Thermocycler

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PCR process

What does 90°Cdo to our

DNA polymerase?

What do you need to do? in tube: DNA, enzyme, primer, nucleotides heat (90°C) DNA to separate strands (denature) cool to hybridize (anneal) & build DNA (extension)

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PCR primers The primers are critical!

need to know a bit of sequence to make proper primers

primers bracket target sequence start with long piece of DNA

& copy a specified shorter segment

primers define section of DNA to be cloned 20-30 cycles

3 steps/cycle30 sec/step

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The polymerase problem

play PCR movie

PCR20-30 cycles3 steps/cycle30 sec/step

Heat DNA to denature it 90°C destroys DNA polymerase have to add new enzyme every cycle

almost impractical!

Need enzyme that can withstand 90°C… Taq polymerase

from hot springs bacteria Thermus aquaticus

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Kary Mullis development of PCR technique

a copying machine for DNA

1985 | 1993

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Biotechnology today: Applications Application of DNA technologies

basic biological research medical diagnostics medical treatment (gene therapy) pharmaceutical production forensics environmental cleanup agricultural applications

…and then there’s the ethics issues!

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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”

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Any Questions??

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What next? After you have cloned & amplified DNA

(genes), you can then tackle more interesting questions how does gene differ from person to

person? …or species to species

is a certain allele associated with a hereditary disorder

in which cells is gene expressed? where is gene in genome?

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