Biotechnology andGenetic Engineering

AP Biology Chapter 20

Terminology

• Genetic engineering – direct manipulation of genetic material for practical purposes

• Biotechnology – use of living organisms or their components to make products for us

• Recombinant DNA – combining pieces of DNA from different organisms

• Gene cloning – making copies of DNA

Making recombinant DNA

• Plasmids (small circular pieces of DNA in bacterial cells) are used to insert pieces of foreign DNA

The DNA is cut using restriction enzymes

What are restriction enzymes?

• Restriction enzymes come from bacteria and recognize a particular pattern of DNA, often 4, 6 or 8 base pairs long, and then cut the DNA within this recognized sequence.

• Bacteria use these enzymes to kill off other competing bacteria by cutting up their DNA.

How do they cut?

STICKY ENDS BLUNT ENDS

ACT GAA TTC CGG AAT GAA TTC

TGA CTT AAG GCC TTA CTT AAG

Where would the enzyme EcoRI cut?

ACT GAA TTC CGG AAT GAA TTC

TGA CTT AAG GCC TTA CTT AAG

There would be three pieces: one 4 bases, one 12 bases, and one 5 bases.

Making recombinant DNA in plasmids

The collection of thousands of clones of bacteria containing recombinant plasmids is called a genomic library.

Genes can be cloned into

vectors such as plasmids

Fig. 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

Fig. 20-2a

DNA of chromosome

Cell containing geneof interest

Gene inserted intoplasmid

Plasmid put intobacterial cell

RecombinantDNA (plasmid)

Recombinantbacterium

Bacterialchromosome

Bacterium

Gene ofinterest

Plasmid

2

1

2

Fig. 20-2b

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

Gene ofInterest

Protein expressedby gene of interest

Basic research andvarious applications

Copies of gene Protein harvested

Basicresearchon gene

Basicresearchon protein

4

Recombinantbacterium

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

3

Steps

1. Plasmid and human DNA are isolated.

2. Both DNAs are cut with the same restriction enzyme.

3. “new” DNA is ligated into plasmid

4. Recombinant plasmids are inserted into bacterial cells.

5. Plate bacteria on agar. Bacteria will express new genes.

Nucleic Acid Hybridization

• Used to detect genes

• The DNA of the cell is denatured to produce single stranded DNA.

• The radioactive probe will hybridize (bond) with complementary bases if present.

• Probes can be radioactive isotopes or

flourescent dyes.

The radioactive probe is made by determining a short segment of the protein sequence, then "back translating" to the possible DNA sequences.  Short DNA sequences are synthesized to match the  protein sequence.  Then these DNA oligomers (known as "oligos") are radiolabeled, and applied to the blotted clones.  They should hybridize only to clones containing sequence encoding the desired protein.

Expression of eukaryoticgenes in prokaryotes

• Use an expression vector with a prokaryotic promoter upstream from the location of the gene.

• Create artificial genes without introns since bacteria do not have the machinery for eliminating introns.

• YACS

What are YACS?

• Yeast artificial chromosomes that carry foreign DNA.

• Yeast cells have plasmids that can act as vectors.

Electroporation

• injecting DNA into eukaryotic cells

PCR Polymerase Chain Reaction

• Used to amplify DNA

• Discovered by Kary Mullis (GT grad)

A Thermocycler

Steps of PCR?

• Denature DNA (94-96 C)

• Anneal (base pair) primers (50 – 65 C)

• Extend primers (72 for polymerase to work)

• Machines called thermocyclers do this.

http://www.dnalc.org/ddnalc/resources/shockwave/pcranwhole.html

• In PCR, a heat-stable DNA polymerase is used, most commonly Taq Polymerase from the thermophilic microbe Thermus aquaticus.  Thomas Brock discovered T. aquaticus  from a hot spring at Yellowstone National Park.

Why is PCR used prior to cloning a gene in cells?

The task of later identifying the clone carrying the gene is simplified.

Applications of PCR

PCR has replaced cloning for many purposes, particularly the sequencing of DNA. 

• It is faster and requires no vectors, which can mutate as they reproduce.  

• It can be used forensically, to amplify tiny amounts of DNA from criminal evidence; or clinically, to detect DNA sequences linked to inherited disorders.  

What is gel electrophoresis?

• A technique to separate DNA based on the movement of DNA fragments from neg to pos (DNA is neg).

• Smaller fragments travel farther.

• Samples are placed in gels.

Gel Electrophoresis

Southern BlottingDNA Fingerprinting

1. Isolate DNA2.Cut DNA into fragments with restriction enzymes.3.Electrophorese.4.Blot onto nylon membrane.5.Apply radioactive probes.6.Wash to remove unbonded probes.7.X-Ray.

Southern Blotting

Sanger Sequencing

• Used to sequence short segments of DNA

• Fragments are incubated with fluorescent dyes.

• When fragments hybridize with the tagged nucleotide, the hybridization stops.

• Fragments are electrophoresed and analyzed.

Early DNA Sequencing

Analyzing Expression of Genes

• Northern Blotting, in situ hybridization – using radioactive probes to look for mRNA being produced

• RT-PCR – Reverse transcriptase-polylmerase chain reaction – makes cDNA from mRNAs and then PCRs the DNA for electrophoresis

• Micro – arrays - Isolate mRNA from cells, make cDNA using reverse transcriptase, then uses cDNA to explore collections of genomic DNA

• Microarrays are useful in discerning gene expression in different tissues AND at different stages of development.

• Different brightness and

colors signify rates of

expression.

Determining Gene Function

• In vitro mutagenesis – changes made to cloned gene, gene returned to cell and it “knocks out” the normal gene. Then look for abnormalities.

• RNA interference (RNAi) – uses double RNA to block translation of mRNA.

Cloning Organisms

• Organismal cloning – producing genetically identical individuals from a single somatic cell of a multicellular organism

In plants• Steward demonstrated

genomic equivalence in plants by growing carrot plants from differentiated root cells.

• Most plant cells remain totipotent, retaining the ability to give rise to a complete new organisms.

In Animals

• Briggs and all transplanted nuclei from embryonic frog cells into enucleated egg cells and produced cloned frogs

• Nuclear transplantation – name of process

• Whether normal development occurred depended on developmental age of the transplanted nucleus.

Fig. 20-17

EXPERIMENT

Less differ-entiated cell

RESULTS

Frog embryo Frog egg cell

UV

Donornucleustrans-planted

Frog tadpole

Enucleated egg cell

Egg with donor nucleus activated to begin

development

Fully differ-entiated(intestinal) cell

Donor nucleus trans-planted

Most developinto tadpoles

Most stop developingbefore tadpole stage

Nuclear Transplantation

And then Dolly came along in 1997

Fig. 20-18

TECHNIQUE

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

Why Dolly died young 6 yrs

• Dolly's telomeres were found to be approximately 80% of the length they should be for a sheep her age.

• Also there is the concern of damaged DNA being carried into the clone

Cloned animals do not look exactly like the transplanted nucleus due to cytoplasmic affects.

RainbowCC

CC and her Surrogate mom

• In most nuclear transplantation studies, only a small percentage of cloned embryos have developed normally to birth

• Many epigenetic changes, such as acetylation of histones or methylation of DNA, must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development

Stem Cells

• Relatively unspecialized cells that continue to reproduce themselves and can be induced to form specialized cells

• Embryonic cells are more totipotent than adult stem cells

• Therapeutic cloning – using stem cells to replace organs and tissues

• Reproductive cloning – using stem cells to reproduce new organisms

• Both raise ethical

debates

Benefits of DNA technology

• Medical Applications

• identification of human genes in which mutation plays a role in genetic diseases

• Single nucleotide polymorphisms (SNPs) are useful genetic markers

• These are single base-pair sites that vary in a population

• When a restriction enzyme is added, SNPs result in DNA fragments with different lengths, or restriction fragment length polymorphisms (RFLP)

Fig. 20-21

Disease-causingallele

DNA

SNP

Normal alleleT

C

Human Gene Therapy

• Gene therapy is the alteration of an afflicted individual’s genes

• Vectors, such as viruses, are used for delivery of genes into specific types of cells, for example bone marrow

• It may be difficult to target cells.• Gene therapy raises ethical questions,

such as whether human germ-line cells should be treated to correct the defect in future generations

Fig. 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

Pharmaceutical Products

• Advances in DNA technology and genetic research are important to the development of new drugs to treat diseases

• In particular “pharm” animals and plants can be used to produce certain products

Fig. 20-23

Forensic Evidence and Genetic Profiles

• An individual’s unique DNA sequence, or genetic profile, can be obtained by analysis of tissue or body fluids

• Even more sensitive is the use of genetic markers called short tandem repeats (STRs), which are variations in the number of repeats of specific DNA sequences

Fig. 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

Environmental Cleanup

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

• Biofuels make use of crops such as corn, soybeans, and cassava to replace fossil fuels

Genetic Engineering in Plants

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

• The Ti plasmid is the most commonly used vector for introducing new genes into plant cells

• Most public concern about possible hazards centers on genetically modified (GM) organisms used as food

• Some are concerned about the creation of “super weeds” from the transfer of genes from GM crops to their wild relatives

Fig. 20-25

Site whererestrictionenzyme cuts

T DNA

Plant with new trait

Tiplasmid

Agrobacterium tumefaciens

DNA withthe geneof interest

RecombinantTi plasmid

TECHNIQUE

RESULTS