uses of dna technology you will need to convince a grant committee to fund further research into...

Uses of DNA technology• You will need to convince a grant committee to

fund further research into your area of application of DNA technology

• Read your assigned section• IN YOUR OWN WORDS, give an argument that

includes 3 points that highlight that your field needs more money than any other

• Identify 2 counterarguments and explain why these may not be valid and you should still receive funding.

One of the key tools in DNA technology is the restriction enzyme

Where do these restriction enzymes come from????

What is their natural function???

How can we use them???

Recombinant DNA

• DNA from 2 sources combined– Can be used to clone genes– Used to produce a particular protein

E. coli bacterium

Plasmid

Bacterialchromosome

Gene of interest

DNA

Cell with DNAcontaining geneof interest

Isolateplasmid

IsolateDNA

1 2

A plasmid is a small circular piece of DNA found in some bacterial cells

Separate from main chromosome

May have genes that give the bacteria an advantage in certain circumstances

Bacteria can take up plasmids from their environment

E. coli bacteriumPlasmid

Bacterialchromosome

Gene of interestDNA


Gene of interest

Isolateplasmid

IsolateDNA

Cut plasmidwith enzyme Cut cell’s DNA

with same enzyme

1

2

34


Bacterialchromosome

Gene of interestDNA


Gene of interest

Isolateplasmid

IsolateDNA

Cut plasmidwith enzyme

Cut cell’s DNAwith same enzyme

1

2

3

4

Combine targeted fragmentand plasmid DNA

5


Bacterialchromosome

Gene of interestDNA


Gene of interest

Isolateplasmid

IsolateDNA

Cut plasmidwith enzyme

Cut cell’s DNAwith same enzyme

1

2

3

4

RecombinantDNAplasmid

Geneof interest

Combine targeted fragmentand plasmid DNA

Add DNA ligase,which closesthe circle withcovalent bonds

5

6


Geneof interest

Recombinantbacterium

Put plasmidinto bacterium

7


Geneof interest


Cloneof cells

Put plasmidinto bacteriumby transformation

Allow bacteriumto reproduce

8

7


Geneof interest


Cloneof cells

Genes or proteinsare isolated from thecloned bacterium

Harvestedproteinsmay be used directly

Examples ofprotein use

Put plasmidinto bacteriumby transformation

Allow bacteriumto reproduce

8

7

Genes may be insertedinto other organisms

Examples ofgene use

9

How is this recombinant DNA made?

Important to use the same restriction enzyme to cut each source of DNA

This allows complementary sticky ends to be created that can later base-pair to combine the DNA

Restriction enzymerecognition sequence

1

2

DNA

Restriction enzymecuts the DNA intofragments

Sticky end


1

2

DNA


Sticky end

3

Addition of a DNAfragment fromanother source


1

2

DNA


Sticky end

3


4

Two (or more)fragments sticktogether bybase-pairing


1

2

DNA


Sticky end

3


4

Two (or more)fragments sticktogether bybase-pairing

DNA ligasepastes the strands

RecombinantDNA molecule5

1. Plasmid DNA is isolated

2. DNA containing the gene of interest is isolated

3. Plasmid DNA is treated with restriction enzyme that cuts in one place, opening the circle

4. DNA with the target gene is treated with the same enzyme and many fragments are produced

5. Plasmid and target DNA are mixed and associate with each other

Copyright © 2009 Pearson Education, Inc.

Steps in cloning a gene

6. Recombinant DNA molecules are produced when DNA ligase joins plasmid and target segments together

7. The recombinant DNA is taken up by a bacterial cell

8. The bacterial cell reproduces to form a clone of cells


Steps in cloning a gene

Use restriction enzymes to break DNA into manageable sized pieces

that we can separate

What can we tell from this?

• It can be used to compare the DNA from different organisms

• Used to detect disease alleles• Used to “match” DNA samples

– Determine parentage– Crime scene forensics

Detecting disease alleles

Problem: if we’re trying to get a bacterium (prokaryote) to make our proteins, they don’t have introns… so, they can’t remove them

Solution: Use reverse transcriptase (found in retroviruses) to make DNA from mature mRNA

PCR is used to amplify DNA sequences

• http://learn.genetics.utah.edu/content/labs/pcr/

– Mix ingredients in a thermocycler• What do you need to make lots of copies of DNA?


Cycle 1yields 2 molecules

21 3

GenomicDNA



3 5 3 5 3 5

Targetsequence

Heat toseparateDNA strands

Cool to allowprimers to formhydrogen bondswith ends oftarget sequences

35

3 5

35

35 35

Primer New DNA

5

DNApolymerase addsnucleotidesto the 3 endof each primer

5


GenomicDNA

3 5 3 5 3 5

Targetsequence

Heat toseparateDNA strands

Cool to allowprimers to formhydrogen bondswith ends oftarget sequences

35

3 5

35

35 35

Primer New DNA

5

DNApolymerase addsnucleotidesto the 3 endof each primer

215

3

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