Restriction Enzyme digestion of DNA - Exercise 8 - PowerPoint Presentation

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Restriction Enzyme digestion of DNA - Exercise 8

Objectives

-Understand how Restriction Enzymes digest DNA.

-Know how to construct a

pAMP

(plasmid) or gel.

-Given the size of fragments, gel, know how to construct a restriction map.

-Given a restriction map know how to construct a gel.

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NOTE: DNA IS Negatively charge

because of the phosphate groups.

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DNA molecules are macromolecules that hold the genetic information of living organisms. They are extremely long, double-stranded polymers of nucleotides.

The covalent bond joining adjacent nucleotides in DNA is called a

phoshodiester

bond.

The

phoshodiester

bonds between nucleotides in DNA molecules are very stable unless they are physically stretched or exposed to enzymes name nucleases.

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Enzymes are capable of breaking (hydrolyzing)

phoshodiester

bonds in DNA molecules. Nucleases can be classified into two major groups:

exonucleases

and

endonuclases

.

Exonucleases

:

If the enzyme digest nucleotides from the ends of the DNA molecules

.

Endonuclases

:

If the enzyme digest nucleotides in the interior of a DNA molecule

.

Restriction

endonuclease

: An

enzythem

that digest DNA by recognizing specific short sequences of bases that are called palindromes.

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A special class of

endonucleases

from a bacteria has been isolated for this experiment. These special enzymes, termed restriction

endonucleases

(RE)

,

digest DNA by breaking bonds only within a specific short sequence of bases. These base sequences usually ran in size from 4-8 base pairs but can be as long as 23 base pairs.

Restriction

endonucleases

confer an adaptive advantage on bacteria by digesting foreign DNA usually from an invading

bacteriphage

(bacterial virus). The resulting DNA fragments can then be further degraded and destroyed by

exonucleases

. These enzymes are used to cut DNA in a precise and predictable manner. They are extensively useful in gene cloning, DNA amplification, and many recombinant DNA technologies.

Restriction

endonuclease

(RE). This RE are also attained from bacteria. In a bacteria where we get these enzymes form there protected because if a virus invades a bacteria cell these

endonuclease

will chop up the virus DNA, its like a defense system, so we can isolate these

endonuclease

for experiments, but bacteria produce these

endonuclease

to protect themselves from foreign DNA entering their cells.

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2 Restriction

Endonucleases

(RE)

EcoR1 &

HindIII

. Both of these recognize different nucleotide sequences.

Each strand of DNA is cut at the

phoshodiester

bond between the G and A bases

(indicated by the arrow signs). Notice that the sequence GAATTC is the same on both strands when each strand is read 5’ -> 3’. Such symmetrical sequences are called

palindromes (In a English language a palindrome reads the same thing in both directions). This enzyme cuts the double strands asymmetrically, leaving protruding ends. These protruding bases are referred to as sticky ends aka compatible cohesive ends.

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EcoR1: EcoR1 recognizes palindrome on DNA, and cuts the bond between G & A, and G & A. When you do that it opens your DNA. For example, if you have plasmid and that palindrome is present once on the plasmid, you’ll get one cut.

If somewhere else that palindrome is present and you incubate it with EcoR1, you’ll get another cut. So every time EcoR1 recognizes this palindrome on your plasmid, it will cut through the DNA. So when it opens up the DNA, may get a couple of unpaired bases, and those unpaired bases are called sticky ends, and if you throw some nucleotides from different species, you can make recombinant DNA.

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Like EcoRI

,

HindIII

also recognizes a

palindromic

sequence, AAGCTT, and produces sticky ends. Sticky ends can hydrogen bond together other because of complementary base pairing.

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Recombinant DNA molecules are compose of DNA fragments from two or more sources. Not all RE’s produce sticky ends. Some enzymes cut DNA to produce blunt ends, as shown here.

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Once the DNA has been digested, the fragments must be separated and identified.

Fragments are separated by

agarose

gel electrophoresis. Agar is a large polysaccharide.

Gel electrophoresis: You put an

agorose

gel (

agrose

is a polysaccharide) and it has spaces, your DNA can move through these spaces, you put a current against this, the negative end is up, the positive is at the bottom, and because your DNA has a negative charge, the

DNA moves down towards the positive end.

Gel is immersed in an ionic buffer. The buffer has a pH above 8.0 DNA at this pH is negatively charge because the phosphates in the DNA backbone have lost hydrogen ions.

The dye molecules serve as the indicator of the

movements of invisible DNA molecule through

the gel as an electric current is run through the gel.

The negatively charged DNA will migrate from the anode to the cathode (negative to positive) alongwith the current.

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Separation of the DNA fragments occurs as they migrate through the network of

agarose

molecules.

Smaller fragments slip through the network fast than large molecules. The rate of migration is a function of fragment size, as well as the density of

agarose

.

The tightness (concentration of

agarose

). High concentration favor smaller fragments.

Low concentration favor large fragments.

Each of migration function of fragment size and density of

agarose

. Depending on what conformation a circular DNA gets, it will run differently in the gel.

So not only does the size of the DNA molecule affect migration rate, but the configuration of the DNA also affects the migration rate

. The DNA that you will electrophoresing can exist in three different conformations.

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1. Supercoil

circular

: 1

st

fastest. When its circular it becomes twisted and turn and be comes a little bit shorter in size. Migrates fastest down the gel. Contains small volume, more compacted.

2.

Linear

: Migrates next fastest down the gel.

3. Nicked (relaxed) circular

: One strand is intact, the other is broken and when it is nicked, it becomes extended. This one is very relaxed and faces the most difficulty making its way through the

agarose

.

Supercoil

< Linear < Nicked (relaxed) circular

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In addition to conformation affecting migration rate, laboratory production of plasmid DNA can be produce very large molecules that migrate very slowly.

Two possible molecules that can be produced are

dimers

and

concatemers

. A

dimer

consists of two plasmids covalently linked in a series end to end. A

Concatemer, for example, might consist of two plasmids with one hooked through the other but not covalently linked to each other. If a purified uncut plasmid is applied to a gel, bands of super coiled plasmid, nicked circular plasmid,

dimers

, and

concatemer

can be observed.

Dimer

: Means that its link together by 2 linksConcatemer: Mean a whole bunch of plasmids linked together but not covalently linked to each other.

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pAMP

- the plasmid DNA. What we did in the experiment on DNA restriction analysis is we took

pAMP

(circle) and incubated the

pAMP

this plasmid with different restriction

endonucleases

.

From your electrophoresis gel, you can estimate the size of

pAMP

.

You can also determine if

pAMP

is circular or linear. Finally, you can use the gel to draw a restriction map. A restriction map is a physical map of a piece of DNA showing recognition sites of specific restriction enzymes separated by lengths marked in numbers of bases. Separated DNA base on size

The pattern of DNA bands is characteristic for a specific DNA sample and the restriction enzymes used to cleave it. A banding pattern can be referred to as a DNA fingerprint. because it is unique to that particular DNA (and the combination of restriction fragments).

We ran a gel to see if we could determine how many DNA fragments you got

. By

electrophoresing

a series of fragments of known size (DNA ladder) along with the DNA samples of interest, the sizes of unknown fragments can be estimated.

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A restriction site is a place where an enzymes cuts DNA, so there are restriction sites for EcoR1, and for

HindIII

.

When constructing the

pAMP

no restriction site where you start and where you finish.

Lane 4: A control to see what uncut plasmid looks like. How uncut DNA traveled whether they made 1 or 2 pieces. It’s your plasmid DNA

DNA

was on tube 4 which acts like a measurements and acts like a ladder. No enzyme (Lane 4).

Lane 5: DNA ladder: DNA digest, containing known base pair lengths compare with fragments in lanes 1-3. You will run DNAs of known size (DNA ladder) to help you estimate the size of your DNA fragments. Lane 5 contains DNAs of known sizes (DNA ladder).

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Prokaryotic (Circular) DNA

DNA from bacteria (both chromosomal DNA and extra chromosomal plasmid DNA) and viruses is often a closed circle. If you have a circular DNA, we know that’s Prokaryotic DNA.

In Prokaryotic DNA, the number of fragments will equal the number of restriction sites.

Eukaryotic (linear) DNA

If you have one restriction site for an enzyme, you would have 2 fragments, and if you have 2 restriction sites for an enzyme, you would have 3 fragments.

In Eukaryotic DNA, the number of fragments is always going to have one more or one less than restriction sites.

In Eukaryotic DNA, there’s no reason to see multiply bands in control lane because Eukaryotic DNA is linear, it doesn’t exist as

supercoil

, relax, or

multimere

so this is a hint in lane 4. So when you have Eukaryotic DNA, you will not see multiply bands in the control lane.

Also, just because they show you multiply bands, not every time your going to have prokaryotic (circular) DNA you get multiply lanes, its only if the DNA has been damaged into a

supercoil

.

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What’s going to effect the movement of the DNA

(Factors)?

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Size: small pieces migrate faster, farther than bigger pieces.

Conformation (shape): Comparing 3 pieces of DNA that are the same size.

Supercoil

< Linear < Nicked (relaxed) circular

Charge: Charge (+,-)DNA is negative because of Phosphate groups (anode) to positive (cathode).

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Digestion of pAMP

with

EcoRI

&

HindIII

We incubated our plasmid under several conditions. Those conditions were that we incubate

pAMP

.

Lane 1: EcoR1 - one bandLane 2: HindIII - one band Lane 3: EcoR1 &

HindIII

- two bands

Lane 4: Water - Our control. We got one main band.

Lane 5: DNA ladder, a tool to measure the size of DNA fragments.

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When constructing the

pAMP

.

There’s no restriction site

where you start and where you

finish the map. You could call

this point the reference point.

Also, all your base pairs

(fragments) have to equal the

total number base pairs

of your

plasmid. For example, 6,000

Bp’s in this example.

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Starting & Ending Point

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Key for pAMP

KEY

Enzyme A: Light green

Enzyme

B: Pink

Enzyme

C: Orange

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Enzyme A

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Enzyme A

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Enzyme A

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Enzyme A

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Enzyme B

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Enzyme B

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Enzyme B

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Enzyme C

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Enzyme C

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Enzyme A + B

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Enzyme A + B

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Enzyme A + B

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Enzyme A + B

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Enzyme A + B

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Enzyme A + B

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Enzyme A + B

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Enzyme A + C

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Enzyme A + C

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Enzyme A + C

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Enzyme A + C

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Enzyme A + C

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Enzyme A + C

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Enzyme B + C

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Enzyme B + C

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Enzyme B + C

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Enzyme B + C

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Enzyme B + C

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Enzyme A + B + C

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Enzyme A + B + C

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Enzyme A + B + C

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Enzyme A + B + C

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Enzyme A + B + C

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Enzyme A + B + C

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Enzyme A + B + C

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Enzyme A + B + C

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Are the number of fragments correct?

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How do you cut and paste DNA?

Enzymes that cut DNA at specific short sequence sites

Restriction enzymes digest DNA

Blunt end cut

Asymmetric end cut

Enzymes that paste complementary DNA fragments together

DNA ligase

Slide66

Using a restriction enzyme and DNA

ligase

to make recombinant DNA

Slide67

Restriction fragment analysis by Southern blotting

Characteristic pattern of bands for each sample

DNA is transferred to paper and denature to single strands

Probe complementary to the DNA sequence of interest

DNA bound to radioactive probe exposes film

Entire genome

Slide68

DNA CLONING AND ITS APPLICATIONS

Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their

plasmids

.

Cloned genes are useful for making copies of a particular gene and producing a gene product.

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RESTRICTION ENZYMES

Restriction enzymes

are essentially molecular scissors that cut DNA at specific nucleotide sequences.

They originate from bacteria and function as a defense system against viral invasion. They “restrict” viral DNA.

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Restriction Enzymes

Cut DNA at highly specific points

Recognize specific sequences

Four to seven bases

Each is unique

Consistent results

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STICKY ENDS

Most restriction enzymes cut double stranded DNA in an asymmetrical fashion.

These cuts leave single stranded nucleotide overhangs that are competent to hydrogen bond.

These overhangs are called “sticky ends”.

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AGAROSE GEL ELECTROPHORESIS

One indirect method of rapidly analyzing and comparing genomes is

gel electrophoresis

.

This technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size.

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Gel Electrophoresis

Separation of DNA fragments

Based on size

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Cathode

Power

source

Anode

Mixture

of DNA

molecules

of differ-

ent sizes

Gel

Glass

plates

Longer

molecules

Shorter

molecules

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Different

Endonucleases

Yield Different Patterns

Taq1 + Pst1

Taq1 + AvaII

E coli

clinical isolates

Slide78

Questions

1. What is a nuclease?

2. How does an

endonuclease

differ from an

exonuclease

?

3. What is a restriction

endonucleases

? Write names of some restriction

endouclease

.

Slide79

Questions

1. What is a nuclease?

DNA held by covalent bond joining adjacent nucleotides in DNA is called a

phosphodiester

bond. The

phosphodiester

bond between nucleotide in DNA molecules are very stable unless they are physically stretched or exposed to enzymes name nucleases.

Enzyme capable of breaking (hydrolyzing)

phosphodiester

bonds in DNA molecules and classified into

exonuclease

and

endonuclease

.

2. How does an

endonuclease

differ from an

exonuclease

?

Endonuclease

digest DNA by breaking

phosphodiester

bonds in the interior of DNA molecule.

Exonuclease

enzyme digest nucleotides from the ends of the DNA molecule.

3. What is a restriction

endonucleases

? Write names of some restriction

endouclease

.

Restriction

endonucleases

are a special class of

Endonuclease

from bacteria to cut DNA.

EcoRI

& Hind III. These are enzymes digest DNA by recognizing specific short sequences of bases called

palindromic

.

Slide80

Questions

4. What are 2 restriction

endonuclease

(RE) that we used in our lab? Write DNA sequences these RE recognize. Do they produce sticky ends or blunt ends when they cut the DNA molecules?

5. How does the number of restriction sites relate to the number of fragments produced for linear DNA or circular DNA?

6. What is

palindromic

DNA sequence?

Slide81

Questions

4. What are 2 restriction

endonuclease

(RE) that we used in our lab? Write DNA sequences these RE recognize. Do they produce sticky ends or blunt ends when they cut the DNA molecules?

EcoRI

& Hind III. Both produce sticky ends when cut.

5. How does the number of restriction sites relate to the number of fragments produced for linear DNA or circular DNA?

Eukaryotic DNA, always going to have one more or one less fragment than you have restriction sites.

Prokaryotic DNA, the number of fragments will equal the number of restriction sites.

6. What is

palindromic

DNA sequence?

Reading from the same thing in both direction to read the sequences bases that restriction

endouclease

recognizes. For example,

M’adam

I’m

adam

.

Slide82

Questions

7. What is electrophoresis? What does

agarose

gel electrophoresis allow us to do?

8. What is the chemical nature of

agarose

?

9. What factors effect the migration rate of DNA through an

agarose

gel?

10. For DNA molecules of equal sizes, how do the different shapes (conformation) of DNA differ in terms of distance traveled through an

agarose

gel?

Slide83

Questions

7. What is electrophoresis? What does

agarose

gel electrophoresis allow us to do?

It’s a gel that allows move fragment of DNA across by attracting DNA, which is negative (anode) to opposite side (cathode) positive side base on size, and conformation of DNA. It will migrate with current.

8. What is the chemical nature of

agarose

?

Polysacchirde

& sea weed.

9. What factors effect the migration rate of DNA through an

agarose

gel?

Size, shape (conformation), and charge.

10. For DNA molecules of equal sizes, how do the different shapes (conformation) of DNA differ in terms of distance traveled through an agarose gel? Supercoil

travels the fastest, follow by linear, & nicked

Slide84

Questions

11. In your

pAMP

electrophoresis experiment, why did you run a DNA ladder (lane 5) and undigested

pAMP

DNA (lane 4)?

12. Write some practical applications for use of restriction end nuclease?

Slide85

Questions

11. In your

pAMP

electrophoresis experiment, why did you run a DNA ladder (lane 5) and undigested

pAMP

DNA (lane 4)?

Lane 4 is control of DNA to see what uncut plasmid looks like.

Lane 5 is DNA ladder: Containing known base pair lengths and use to compare with fragments in lanes 1-3.

12. Write some practical applications for use of restriction end nuclease?

LOOK AT SLIDES 85-107 on this presentation.

Slide86

Applications of DNA Technology

Diagnosis of disease

Viral genome detection (HIV)

Genetic disorders (screen for defective genes – hemophilia, CF, breast cancer)

Production of pharmaceutical products

Insulin for diabetes

Gene Therapy

Replace or supplement of a defective gene

DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products.

An example of DNA technology is the microarray, a measurement of gene expression of thousands of different genes.

Slide87

Manipulation of DNA

Selective breeding

Domesticated animals

Dogs

Corn

Molecular Approaches

Power, precision and speed

Transfer of one gene

Transfer between species

Cloning of DNA

-Restriction

endonucleases

-Vector

-Gel electrophoresis

-PCR

Uses of DNA technology

-GMO

-Human Disease

-DNA Fingerprinting

-Bioremediation

Bioremediation

-Biological methods dealing with pollution, oil spills, pesticide residues.

-Gene responsible for breakup of harmful products (enzyme) cloned into bacteria.

-Bacteria are seeded into a contaminated area.

Slide88

Other applications…

Environmental Uses

Mining minerals

Detoxifying wastes (oil, sewage, pollution)

Agricultural Uses

Transgenic organisms

Sheep with better wool

Pig with leaner meat

Genetic engineering in plants

Resistant to disease and spoilage

Delayed ripening

Forensic Investigation

Identifying criminal by DNA fingerprinting

Paternity tests

Slide89

Therapeutic Cloning

Therapeutic Cloning

Creates embryonic stem cells

Produces material for organ transplants

Has been challenged on ethical grounds

Slide90

Reproductive Cloning

Reproductive Cloning

Creates living child

Produces offspring identical to parents

Has been done in animals, not people

Slide91

Gene Therapy

Slide92

DNA Fingerprinting

Identifies individuals

Disease prevalence

Forensics

Paternity

RFLP analysis

PCR amplification

Slide93

Sickle Cell RFLP

Slide94

94

Detection of Sickle-Cell

Applications: Detecting mutations

Slide95

RFLP – Restriction Fragment Length Polymorphism

DNA cut with Restriction Enzyme

Gel electrophoresis

DNA hybridization

Compare bands

Applications: Catching the bad guys

DNA fingerprinting

 

-Cut DNA with Restriction Enzymes

-Gel electrophoresis

-Compare bands

Slide96

Figure 20.17 DNA fingerprints from a murder case

PCR amplify small amounts of DNA from crime scene

Digest DNA and compare pattern of bands – DNA fingerprint

Slide97

MEDICAL APPLICATIONS

One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases.

We don’t really understand a genetic disease until we know the mutation, how the gene works, and how the protein product functions both normally and in the disease state.

Slide98

HUMAN GENE THERAPY

Gene therapy

is the alteration of an afflicted individual’s genes.

Gene therapy

holds great potential for treating disorders traceable to a single defective gene.

Vectors

are used for delivery of genes into cells.

Gene therapy

raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations.

Slide99

PHARMACEUTICAL PRODUCTS

Some pharmaceutical applications of DNA biotechnology:

Large-scale production of human hormones and other proteins with therapeutic uses

Production of safer vaccines

Slide100

SOME EXAMPLES OF BIOTECHNOLOGY PRODUCTS

Tissue Plasminogen Activator- dissolves bloodclots.

Human growth hormone.

Insulin

Blood clotting factor VIII.

Recombinant vaccines such as for Hepatitis B.

Bovine Growth Hormone.

Tissue Growth Factor beta.

Platelet Derived Growth Factor.

Slide101

FORENSIC EVIDENCE

DNA “fingerprints

” obtained by analysis of tissue or body fluids can provide evidence in criminal and paternity cases.

A

DNA fingerprint

is a specific pattern of bands of RFLP markers on a gel.

The probability that two people who are not identical twins have the same DNA fingerprint is very small.

Exact probability depends on the number of markers and their frequency in the population.

Slide102

SOME UNUSUAL PLACES FORENSIC SCIENTISTS LOOK FOR DNA EVIDENCE.

Slide103

DNA FINGERPRINTS CAN BE USED TO DETERMINE PATERNITY

Slide104

ENVIRONMENTAL CLEANUP

Genetic engineering

can be used to modify the metabolism of microorganisms.

Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials.

Slide105

AGRICULTURAL APPLICATIONS

DNA technology is being used to improve agricultural productivity and food quality.

Slide106

ANIMAL HUSBANDRY AND “PHARM” ANIMALS

Transgenic organisms

are made by introducing genes from one species into the genome of another organism.

Transgenic animals

may be created to exploit the attributes of new genes (such as genes for faster growth or larger muscles).

Other

transgenic organisms

are pharmaceutical “factories,” producers of large amounts of otherwise rare substances for medical use.

Slide107

GENETIC ENGINEERING

IN PLANTS

Agricultural scientists have endowed a number of crop plants with genes for desirable traits.

Herbicide resistance.

Resistance to pests and disease.

Improved nutrition.

Slide108

GOLDEN RICE

Genetically modified to accumulate beta carotene (vitamin A).

Over a million children a year go blind from vitamin A deficiency.

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