Chapter 13 – Recombinant DNA and Genetic

Engineering

College Prep Biology Mr. Martino

Introduction• Gene Therapy: transfer of one or more

modified genes into an individual’s cells – Correct genetic defect– Boost immune system

• Recombinant DNA Technology: science of cutting and recombining DNA from different species– Genes are then placed into bacterial, yeast or

mammalian cells and replicated

• Genetic Engineering: genes are isolated, modified, and inserted back into a cell– also called biotechnology

15.1 Making Recombinant DNA

• Restriction enzyme: enzyme that chops up DNA at a specific sequence– Bacterial– Viral defense mechanism– May cut a DNA strand a

few times– Helpful in studying DNA– Produces “sticky ends”

which may pair with other DNA

• Genome: all the DNA in a haploid number of chromosomes for each species

• Plasmids: small circle of DNA– In bacterial cells– Insert foreign DNA (gene) into

and put back in bacteria – reproduces naturally making a DNA clone

– Cloning vector: plasmid used to accept foreign DNA and replicate it

• Reverse transcriptase: enzyme from RNA viruses that perform transcription in reverse (RNA to DNA)– cDNA: (copied DNA) mature

mRNA transcript that has already been spliced • Bacteria cannot remove

introns and splice exons• Reverse transcriptase makes

DNA from mRNA to insert into plasmid

15.2 PCR – Polymerase Chain Reaction

• PCR: a fast method of amplifying (making lots of copies) DNA– DNA isolated, mixed with

DNA polymerase, nucleotides, and some other good stuff• Produces 2 daughters• Daughters replicate, etc.• 1 DNA molecule generates

100 billion in a few hours– Used in evolution research,

analyze DNA from fossils, analyze embryos, court cases

15.3 DNA Fingerprints• No two people have

exactly identical DNA– Except identical twins

• DNA Fingerprint: unique set of DNA fragments– Used to determine

paternity, solve crimes, etc.

– 99.9% all human DNA is identical

– Focus on highly variable areas of tandem repeats• Mutations occur within

families and are more common in these areas

• Gel electrophoresis: uses an electric current to force DNA fragments through a gel– DNA is negative– Size of fragment

determines how far it migrates• The fewer tandem

repeats the farther it travels

• Differences in homologous DNA sequences resulting in fragments of different lengths are restriction fragment length polymorphisms (RFLP’s)

15.4 DNA Sequencing• 1995 – entire DNA

sequence for a bacterium was determined

• 4/25/03 – Human genome completed

• Several bacteria, yeast, Drosophila,C. elegans - worm, Arabidopsis - weed, Mickey…a mouse, just completed 3/31/04 – a rat)

• Used a sequencing machine

15.5 Isolating Genes• Genomic Library: set of

DNA fragments from an organism’s genome

• Complementary RNA sequence can be synthesized with a radioactive isotope tag called a probe– Used to find a specific

gene– Tags the gene

whenever encountered– Gene may then be

isolated

15.6 Using the Technology

• True human insulin is now manufactured

• Also somatotropin (growth hormone), blood-clotting factors, hemoglobin, interferons (cancer research), and various other drugs and vaccines

• Bacteria for oil spill clean up and other environmental pollution

15.7 Designer Plants• Genetically engineered

plants have been developed for pharmaceuticals, herbicide, pest, and disease resistance, larger and tastier plants, fruits, and vegetables with greater yields– Corn, cotton, potato, soy

bean, etc

15.8 Gene Transfers in Animals

• Cloning holds promises for future– Clone organs and

tissues– Possibly modify

animals to be more disease resistant and produce greater quantities of products

– Not currently occurring in farm animals

• 1997 – the first animal was cloned – Dolly a lamb– 1. Remove nucleus

from cell– 2. Transfer nuclei from

desired cells into unfertilized eggs

– 3. Implant the “zygote” into surrogate mother

– Since Dolly – we have cloned mice, rats, cows, cats, mules, horses, and Rhesus monkeys along with a couple of endangered animals

Human Genome• HGP – an int’l effort to map and

sequence all human genes– 15 countries started 11/1/90

and finished 4/25/03 (50 years after Watson & Crick paper published)

– 1. Genome – only 30,000 genes so it took less time• Includes mapping &

sequencing of other species for comparison

– 2. RNA transcription – more difficult since 30,000 genes code for 80,000 proteins due to alternative splicing

– 3. Proteome – quest for every human protein

15.9 Who Gets Enhanced?• HGP already has an ethics committee due to

insistence of James Watson– HGP needs to be used to help people and

must be regulated by laws– Must prevent invasion of privacy and

discrimination by insurance companies, employers,etc.

– Must prevent Eugenics: purging of “undesirable” traits from human population (Hitler)

– Science provides society with knowledge and opportunities – society requires rules and constraints to prevent abuse