FRONTIERS OF BIOTECHNOLOGY

MANIPULATING DNA• Scientists use several techniques to

manipulate DNA

RESTRICTION ENZYMES CUT DNA• Why cut DNA?– To study specific genes

instead of ALL the genes on a chromosome

• Restriction enzymes act as molecular scissors– Recognize specific

sequences• Some leave “blunt ends”• Some leave “sticky ends”

RESTRICTION MAPS SHOW THE LENGTHS OF DNA FRAGMENTS

• Gel Electrophoresis: a technique that uses an electrical field within a gel to separate molecules by their size

• DNA is negatively charged and moves toward the positive pole when the electrical field is applied

• Smallest DNA fragments move the fastest

• A pattern of bands is formed

GEL ELECTROPHORESIS

POLYMERASE CHAIN REACTION• PCR: technique

that produces millions of copies of a specific DNA sequence in just a few hours

• Invented by Kary Mullis in 1983

PCR• Uses: – DNA to be copied– DNA polymerase– Plenty of nucleotides A, T, C, and G– Two primers

• 3 Step Process:– Separating– Binding– Copying

RFLPs• Restriction Fragment Length Polymorphisms

• No two individuals have the same genetic material except identical twins

• Restriction enzymes cut at different places, depending on the DNA sequence

• The lengths of DNA restriction fragments are different between two individuals

DNA FINGERPRINTING• A DNA fingerprint is a type of

restriction map

• Representation of parts of a individual’s DNA that can be used to identify a person at the molecular level

• Focuses on noncoding regions of DNA, or DNA sequences outside genes

DNA FINGERPRINTING• DNA sample from:

– Blood– Semen– Bone – Hair

• …Useful in forensics!

DNA FINGERPRINTING IS USED FOR IDENTIFICATION

• DNA fingerprints and probability– Compare at least 5 regions of the

genome

GENETIC ENGINEERING• Entire organisms can be cloned

• Clone: genetically identical copy of a gene or of an organism

• New genes can be added to an organism’s DNA

4 BASIC STEPS TO GENETIC ENGINEERING

• 1. Cutting DNA

• 2. Making recombinant DNA

• 3. Cloning

• 4. Screening

STEP 1: CUTTING DNA

• The DNA from the original organism containing the gene of interest is cut by restriction enzymes

• Restriction Enzymes: bacterial enzymes that destroys foreign DNA molecules by cutting them at specific sites

STEP 1: CUTTING DNA

• Vector: Any agent, such as a plasmid, that carries the gene of interest into another cell

• Plasmid: A circular DNA molecule that is usually found in bacteria and that can replicate independent of the main chromosome

RECOMBINANT DNA

• DNA molecules that are artificially created

• HOW?????

• Created by combining DNA from different sources

EXAMPLE: INSULIN• A protein hormone that controls sugar

metabolism

• Diabetics cannot produce enough

• Must take doses of insulin daily

• Before genetic engineering, insulin was extracted from the pancreases of slaughtered cows and pigs and then purified

• Today the human insulin gene is transferred to bacteria through genetic engineering

• Because the genetic code is universal, bacteria can transcribe and translate the human insulin gene

STEP 2: MAKING RECOMBINANT DNA

• DNA fragments from the gene of interest are combined with the DNA fragments from the vector

• DNA ligase: an enzyme that bonds the DNA fragments together

• The host cell then takes up the recombinant DNA

STEP 3: CLONING

• Gene Cloning: many copies of the gene of interest are made each time the host cell reproduces

• Remember: bacteria reproduce by binary fission, producing identical offspring with the plasmid DNA!

STEP 4: SCREENING• Cells that have received the particular gene

are separated from the cells that did not take up the vector with the gene of interest

• The cells can transcribe and translate the gene of interest to make the protein coded for the gene

CONFIRMATION OF A CLONED GENE

• Southern Blot: a technique used to test for the presence of a specific gene

NORTHERN BLOT• Similar to a

Southern Blot

• Uses RNA instead of DNA

GENETIC ENGINEERING PRODUCES ORGANISMS WITH

NEW TRAITS

SELECTIVE BREEDING• Allowing only those animals with

desired characteristics to produce the next generation

• Horses, cats, farm animals, crops

HYBRIDIZATION

• Crossing dissimilar individuals to bring together the best of both organisms

• Hybrids: the individuals produced from such crosses

• For example, a disease resistant plant and the food producing capacity of another

INBREEDING• The continued breeding of individuals with

similar characteristics

• Often seen in dogs

• Retains characteristics but has risks

• Genetically similar individuals could bring together two recessive alleles for a genetic defect

TODAY…GENETIC ENGINEERING

GENETICALLY ENGINEERED CROPS• More tolerant to

drought

• Plants that can adapt to different soils, climates, and environmental stresses

GENETICALLY ENGINEERED CROPS• Resistant to

biodegradable weedkiller Glyphosate (kills weeds but now doesn’t kill the crop)

• Resistant to insects (gene injures the gut of chewing insects)-therefore plant doesn’t need to be sprayed with pesticides

MORE NUTRITIOUS CROPS• Improve the nutritious value

of many crops

• Asia: rice is a staple food

• Low in iron an beta carotene

• Iron deficient and poor vision

• Genetic engineers have added genes to rice from other plants to overcome this deficiency

POTENTIAL PROBLEMS TO GM CROPS

• Concern that some weeds will become resistant to the weed killer Glyphosate

• New weed-control alternatives will have to be implemented

POTENTIAL PROBLEMS TO GM CROPS

• Nutritional value has been increased in many crops

• Crops must be tested to make sure consumers are not allergic to the GM product

GENE TECHNOLOGY: ANIMAL FARMING

• Farmers added growth hormones to the diet of cows to increase milk production

• Growth hormone was extracted from the brains of dead cows

• The hormone was introduced into bacteria and added as a supplement to a cow’s diet

TRANSGENIC ANIMALS• Animals that have

foreign DNA in their cells

• Human genes have been added to farm animals in order to get the farm animals to produce human proteins in their milk

TRANSGENIC ANIMALS• This is complex and cannot be made by

bacteria through gene technology

• Human proteins are extracted from the animal’s milk and sold for pharmaceutical purposes

• Cloning animals: creating herds of identical animals that can make medically useful proteins

CLONING FROM ADULT ANIMALS• The intact nucleus of an embryonic or fetal

cell (whose DNA has been recombined with a human gene) is placed into an egg whose nucleus has been removed

• The egg with the new nucleus is put in the uterus of a surrogate, or substitute mother and allowed to develop

CLONING FROM ADULT ANIMALS

• 1997 Ian Wilmut first successful cloning using differentiated cells from an adult animal

• Dolly the sheep

CLONING FROM ADULT ANIMALS• Differentiated cells: cells that

have become specialized to become specific cell types

• Scientists had thought that embryonic or fetal cells were the only way…wrong!

CLONING FROM ADULT ANIMALS• Mammary cells from one sheep were fused

with egg cells without nuclei form a different sheep

• The fused cells divided to form embryos, implanted into surrogate mothers

• Only one survived the cloning process

• Dolly, identical to the sheep that provided the mammary cell

PROBLEMS WITH CLONING

• Only a few of the cloned offspring survive for long

• Many become fatally oversized

• Problems in development

GENOMIC IMPRINTING• The right combination of genes are

turned “on” and “off” during early development

• The egg takes years to develop the genomic imprint

• In cloning, the egg divides within minutes

GENOMIC IMPRINTING

• Reprogramming is not possible in such a short time

• Critical errors in development can occur

• Because of these technical problems and ethical problems, cloning humans is illegal in most countries

CONCERNS ABOUT GENETIC ENGINEERING

• Ethical?

• GM crops

– Not enough research had been done to see if added genes might cause allergic reactions or have other unknown side effects

– Interbreeding with natural plants…what does it mean?

GENOMICS INVOLVES THE STUDY OF GENES, GENE FUNCTIONS, AND ENTIRE

GENOMES

• Genomics: The study of genomes, which can include the sequencing of all of an organism’s DNA

• Gene sequencing: determining the order of DNA nucleotides in genes or in genomes

THE GEOGRAPHY OF THE GENOME

• Only 1-1.5% of the human genome codes for proteins

• Each human cell contains about 6 feet of DNA

• Less than 1 inch are exons

•

THE GEOGRAPHY OF THE GENOME

• Human cells contain about 25,000 genes (scientists had expected 120,000!)

• Only 2x the number of genes in a fruit fly!

• Many human genes are identical to those of other species

• All humans are genetically close (DNA of any 2 people is 99.9% identical)

THE HUMAN GENOME PROJECT• Our genome is relatively small! 3 billion base

pairs, but only between 30,000-40,000 genes

• Project started in 1990 with 2 main goals:

– Map and sequence all of the DNA base pairs of the human chromosomes (accomplished in 2003)

– Identify all of the genes within the sequence (still be worked on)

THE HUMAN MICROBIOME PROJECT• 200 scientists at 80 institutions

sequenced the genetic material of bacteria taken from nearly 250 healthy people

• As many as a thousand bacterial strains on each person.

• Each person’s collection of microbes, the “microbiome”, was unique

TECHNOLOGY ALLOWS THE STUDY AND COMPARISON OF BOTH GENES AND

PROTEINS• Bioinformatics: the use of computer databases

to organize and analyze biological data

• DNA microarrays: tools that allow scientists to study many genes, and their expression at once; a small chip dotted with the genes being studied

• Proteomics: the study and comparison of all the proteins that result from an organism’s genome

GENETIC SCREENING AND GENE THERAPY

• Genetic screening: the process of testing DNA to determine a person’s risk of having or passing on a genetic disorder

• Gene therapy: the replacement of a defective of missing gene, or the addition of a new gene, into a person’s genome to treat a gene

GENETICALLY ENGINEERED DRUGS & VACCINES

• Possibilities for the applications of genetic engineering are endless!

DRUGS• Many genetic disorders and other human

illnesses occur when the body fails to make critical proteins

• Example: juvenile diabetes– Body is unable to control levels of sugar in the blood

because the protein insulin cannot be made

• Example: Hemophilia– Factor VIII, a protein that promotes blood clotting– Donated blood was sometimes infected with HIV

and hepatitis B– Genetically engineered factor VIII eliminates these

risks

VACCINESTraditional Vaccines

• Many viral diseases, such as smallpox and polio, cannot be treated effectively by existing drugs

• Vaccine: a solution containing all or part of a harmless version of a pathogen (disease-causing microorganism)

• When the vaccine is injected, the immune system recognizes the pathogen’s surface proteins and responds by making defensive proteins called antibodies

Genetically Engineered Vaccines• Avoid the danger of giving a

patient a disease

• The genes that encode the pathogen’s surface proteins can be inserted into the DNA of harmless viruses, such as cowpox

• The modified, harmless cowpox virus becomes an effective, safe vaccine