BiotechnologyBiotechnologyChapter 20Chapter 20

Gene technologyGene technology

BiotechnologyBiotechnology• Manipulation of organisms to make

useful products

Genetic engineeringGenetic engineering• Manipulation of genes • Gene cloning: • Multiple copies of a single gene• Produce a specific product

Fig. 20-2Fig. 20-2

DNA of chromosome

Cell containing geneof interest

Gene inserted intoplasmid

Plasmid put intobacterial cell

RecombinantDNA (plasmid)

Recombinantbacterium

Bacterialchromosome

Bacterium

Gene ofinterest

Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest

Plasmid

Gene ofInterest

Protein expressedby gene of interest

Basic research andvarious applications

Copies of gene Protein harvested

Basicresearchon gene

Basicresearchon protein

Gene for pest resistance inserted into plants

Gene used to alter bacteria for cleaning up toxic waste

Protein dissolvesblood clots in heartattack therapy

Human growth hor-mone treats stuntedgrowth

2

4

1

3

Recombinant DNARecombinant DNA• 1970’s• Combining genes from different

sources• Even different species• Combined into single DNA• Example: Bacteria and mammal

Recombinant DNARecombinant DNA• Genetically modified bacteria • Mass produce beneficial chemicals• Insulin• Growth hormone• Cancer drugs• Pesticides

PlasmidPlasmid

PlasmidPlasmid• Small separate circular DNA• Replicated same as main DNA• Foreign DNA added to plasmid • Replicated along with plasmid

Recombinant DNARecombinant DNA• Nucleases:• Enzymes that degrade DNA• Restriction endonulceases: • Restriction enzymes• Cut DNA into fragments at specific

points

Recombinant DNARecombinant DNA• Restriction sites: • Places where DNA is cut• Short DNA sequence

Recombinant DNARecombinant DNA• Restriction enzyme recognizes short

sequences in DNA• Cuts at these sequences• Staggered cut• Leaves single-stranded ends• Called “sticky ends”

D:\Chapter_20\A_PowerPoint_Lectures\20_Lecture_Presentation\2003RestrictionEnzymesA.html

                                         

               

Recombinant DNARecombinant DNA• Sticky ends enables other DNA to join• DNA fragments from other sources• Match ends by base pairs

(complementary sequences)• DNA ligase:• Enzyme combines ends• Forms a phosphodiester bond

                                       

                 

                                       

                 

Recombinant DNA (Process)Recombinant DNA (Process)• 1. Isolate gene of interest & bacterial

plasmid• 2. Cut DNA & plasmid into fragments• 3. Mix DNA fragments with cut plasmid. • Fragment with gene of interest is

inserted into the plasmid• 4. Recombinant plasmid is mixed with

bacteria

Recombinant DNA (Process)Recombinant DNA (Process)• 5. Bacteria with recombinant DNA

reproduce• 6. Isolate bacterial clones that

contain gene of interest • Producing protein of interest • 7. Grow large quantities of bacteria

that produce the protein D:\Chapter_20\A_PowerPoint_Lectures\20_Lecture_Presentation\2004CloningAGeneA.html

Fig. 20-4-4Fig. 20-4-4

Bacterial cell

Bacterial plasmid

lacZ gene

Hummingbird cell

Gene of interest

Hummingbird DNA fragments

Restrictionsite

Stickyends

ampR gene

TECHNIQUE

Recombinant plasmids

Nonrecombinant plasmid

Bacteria carryingplasmids

RESULTS

Colony carrying non-recombinant plasmidwith intact lacZ gene

One of manybacterialclones

Colony carrying recombinant plasmid with disrupted lacZ gene

Recombinant DNARecombinant DNA• Vector:• DNA molecule that carry foreign DNA• Enters & replicates in the host• Plasmids & phages are common

vectors• Phages are larger than plasmid • Can handle inserts up to 40 kilobases

PCRPCR• Polymerase chain reaction• Amplify DNA• Makes large quantities of DNA• 1985

PCRPCR• Heated• Denatured • DNA primer• Heat stable DNA polymerase• Makes DNA

Fig. 20-8Fig. 20-85

Genomic DNA

TECHNIQUE

Cycle 1yields

2molecules

Denaturation

Annealing

Extension

Cycle 2yields

4molecules

Cycle 3yields 8

molecules;2 molecules

(in whiteboxes)

match targetsequence

Targetsequence

Primers

Newnucleo-tides

3

3

3

3

5

5

51

2

3

Gel electrophoresis Gel electrophoresis • Study DNA• Polymer (gel)• Restriction fragments• Separates DNA based on charge &

size• Nucleic acids negative charge

(Phosphates)• Migrate towards + end (red)

Fig. 20-9Fig. 20-9

Mixture ofDNA mol-ecules ofdifferentsizes

Powersource

Powersource

Longermolecules

Shortermolecules

Gel

AnodeCathode

TECHNIQUE

RESULTS

1

2

+

+

–

–

Fig. 20-10Fig. 20-10

Normalallele

Sickle-cellallele

Largefragment

(b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

201 bp175 bp

376 bp

(a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene

Normal -globin allele

Sickle-cell mutant -globin allele

DdeI

Large fragment

Large fragment

376 bp

201 bp175 bp

DdeIDdeI

DdeI DdeI DdeI DdeI

Cloning Cloning • Multicellular organisms come from a

single cell.• Offspring are identical

Cloning Cloning • 1950• Carrots• Totipotent: • Mature cells that undifferentiated• Give rise to any type of cells• Common in plants

Cloning Cloning • Nuclear transplantation• Nucleus of an unfertilized/fertilized

egg is removed• Replaced with nucleus of

differentiated cell• Direct development of cell into

tissues etc.

Cloning Cloning • Removed nuclei from an egg• Mammary cells • Fused with egg cells• Dolly, 1997, identical to mammary

cell donor• Died prematurely age 6• Arthritis & lung disease

Fig. 20-18Fig. 20-18TECHNIQUE

Mammarycell donor

RESULTS

Surrogatemother

Nucleus frommammary cell

Culturedmammary cells

Implantedin uterusof a thirdsheep

Early embryo

Nucleusremoved

Egg celldonor

Embryonicdevelopment Lamb (“Dolly”)

genetically identical tomammary cell donor

Egg cellfrom ovary

Cells fused

Grown inculture

1

33

4

5

6

2

Fig. 20-19Fig. 20-19

Cloning Cloning • Few develop normally• Abnormalities• Epigenetic changes to the

chromatin• More methylation of chromatin• Reprogram chromatin of

differentiated cell

Stem cellsStem cells• Started 1998 at UW• Early embryonic cells• Potential to become any type of cell• Master cell generates specialized

cells• Such as muscle cells, bone cells, or

blood cells

Stem cellsStem cells• Embryos• Bone marrow• Umbilical cord blood• Blood stem cells• ?? Turn skin cells into embryonic

stem cells• Therapeutic cloning

Fig. 20-20Fig. 20-20

Culturedstem cells

Early human embryoat blastocyst stage

(mammalian equiva-lent of blastula)

Differentcultureconditions

Differenttypes ofdifferentiatedcells

Blood cellsNerve cellsLiver cells

Cells generatingall embryoniccell types

Adult stem cells

Cells generatingsome cell types

Embryonic stem cells

From bone marrowin this example

Medical applicationsMedical applications• Genetic markers• Detect abnormal disease• SNP• Single nucleotide polymorphisms• Single base pair site where variation

is found• RFLP• Restriction fragment length

polymorphisms

Fig. 20-21Fig. 20-21

Disease-causingallele

DNA

SNP

Normal alleleT

C

Medical applicationsMedical applications• Gene therapy• Treat genetic defects• Alters person’s genes• 2 girls with rare blood disease• CF (vectors are viruses)• SCID (immune disorder)• Injected viral DNA with normal gene

Fig. 20-22Fig. 20-22

Bonemarrow

Clonedgene

Bonemarrowcell frompatient

Insert RNA version of normal alleleinto retrovirus.

Retroviruscapsid

Viral RNA

Let retrovirus infect bone marrow cellsthat have been removed from thepatient and cultured.

Viral DNA carrying the normalallele inserts into chromosome.

Inject engineeredcells into patient.

1

2

3

4

Medical applicationsMedical applications• Transgenic animal• Gene from one animal is inserted

into another• Goat milk protein anti-thrombin• Isolated from milk• “pharm” animals

AnimalsAnimals• Transgenic animals engineered for

specific traits • Genetically create a racehorse• Not have to breed• Sheep with better wool??

Agricultural applicationsAgricultural applications• Manipulate tomatoes • Do not ripen as fast• “Flavr-Savr”• Slows down ethylene production

Agricultural applicationsAgricultural applications• Introduce genes to plants• Enable them to “fix” nitrogen

• Convert N2 to NH3

• Help eliminate use fertilizers• Cut $$

Agricultural applicationsAgricultural applications• Herbicide resistance• Plant genetically resists the herbicide• Insect resistance

Agricultural applicationsAgricultural applications• Transgenic rice• “golden rice”• Rice with genes that code for better

absorption of iron and beta carotene• First of many genetically engineered

foods• Helps dietary deficiencies

ForensicsForensics• Genetic profile:• Individual genetic markers• “DNA fingerprint”• RFLP• STR• Short tandem repeats• Occur in specific regions in genome• Unique

Fig. 20-24Fig. 20-24This photo shows EarlWashington just before his release in 2001,after 17 years in prison.

These and other STR data exonerated Washington andled Tinsley to plead guilty to the murder.

(a)

Semen on victim

Earl Washington

Source of sample

Kenneth Tinsley

STRmarker 1

STRmarker 2

STRmarker 3

(b)

17, 19

16, 18

17, 19

13, 16 12, 12

14, 15 11, 12

13, 16 12, 12

Concerns over genetic Concerns over genetic engineeringengineering

• Genetically modified foods• Harmful?• Genetically engineered gametes • Blonde and blue eyes??