Genetic Engineering Biotechnology 2006 2007 Genetic Engineering Process

Genetic Engineering Biotechnology 2006 -2007

Genetic Engineering • Process of Manipulating & Using DNA

Genetic Engineering: Isolating & Using Genes • Manipulating DNA – A. • 1. • 2. • 3. – B. • 1. • 2. • 3. The Tools of Molecular Biology DNA Extraction Cutting DNA Separating DNA Using the DNA Sequence Reading the Sequence Cutting and Pasting Making Copies

Think About it… • All living things have the same genetic material (DNA) and use the same genetic code. • Gene sequences (instructions for making a protein) can be “read” and used in the same way by and all living things • Biologists decided that, if given the technology, genes for desirable traits could be transferred from one organism to another

The code is universal • Since all living organisms… – use the same DNA – use the same code book – read their genes the same way

Can we mix genes from one creature to another? YES!

A Brave New World

How do we do mix genes? • Genetic engineering – find gene – cut DNA in both organisms – paste gene from one creature into other creature’s DNA – insert new chromosome into organism – organism copies new gene as if it were its own – organism reads gene as if it were its own – organism produces NEW protein: Remember: we all use the same genetic code!

Restriction Enzymes… “little Molecular Scissors” • Restriction enzymes – “recognize” unique nitrogen-base sequences – Called “recognition sites” – different restriction enzymes “recognize” different nitrogen-based sequences – Enzymes “CUT” DNA strands at specific locations – DNA fragments now have uneven “sticky ends” (unpaired bases) – “Sticky ends” allow the DNA fragment to splice (base –pair) into another open DNA strand

Cutting DNA • DNA “scissors” – enzymes that cut DNA – restriction enzymes • used by bacteria to cut up DNA of attacking viruses • Eco. RI, Hind. III, Bam. HI – cut DNA at specific sites • enzymes look for specific base sequences GTAACGAATTCACGCTT CATTGCTTAAGTGCGAA

Original DNA Restriction Enzymes Recognition sequences strand DNA sequence Restriction enzyme Eco. RI cuts the DNA into fragments. Sticky end Restriction enzyme cuts both strands of the DNA Between the A and G following a CTTAAG sequence

Figure 14 -18 DNA Fingerprinting Once the DNA is Cut, the “repeat” fragments are Labeled using Section 14 -3 radioactive probes allowing us to see bands Following Electrophoresis Restriction enzyme Chromosomes contain large amounts of DNA called repeats that do not code for proteins. This DNA varies from person to person. Here, one sample has 12 repeats between genes A and B, while the second sample has 9 repeats. Go to Section: Restriction enzymes are used to cut the DNA into fragments containing genes and repeats. Note that the repeat fragments from these two samples are of different lengths. The DNA fragments are separated according to size using gel electrophoresis. The fragments containing repeats are then labeled using radioactive probes. This produces a series of bands—the DNA fingerprint.

Electrophoresis Lab • /

Gel Electrophoresis used to separate the fragments by Size And Charge DNA plus restriction enzyme Apply an Electric Field Power source Longer fragments Mixture of DNA fragments Gel Shorter fragments An electric current causes the fragments to move from the well toward The Positive end of the field; shorter fragments migrate farther and faster Than the larger fragments

DNA fingerprint • Genes form banding patterns • No two banding patterns are the same • Called DNA fingerprints – Used in identifying remains – Crime investigations – Next of kin

Figure 13 -8 isolated, PCR Once the genes are we can make Extra copies through a cloning process Section 13 -2 Called PCR (polymerase Chain Reaction) DNA polymerase adds complementary strand 1 st DNA heated to separate strands DNA fragment to be copied Go to Section: PCR cycles 1 2 3 4 5 etc. DNA copies 1 2 4 8 16 etc.

Sneaking In • You probably have heard of computer viruses. • Once inside a computer, these programs follow their original instructions and override instructions already in the host computer. • Scientists use small “packages” of DNA to sneak a new gene into a cell, much as a computer virus sneaks into a computer.

VECTORS …. Deliver the genes • Vectors are used to deliver DNA (genes) into another organism’s cells • Vectors can be either: – Plasmids (circular piece of bacterial DNA) – Viruses “nonliving” particles that contain a strand of DNA

“Prepping” the Vectors • Recall that restriction enzymes are used to cut-out the gene we want to use • “Sticky ends” are created (unpaired bases) • The SAME restriction enzyme is used to cut and open-up the DNA of the Plasmid or the Virus • “Sticky ends” are created in vector’s DNA too

Splicing the gene into the Vector • When the “sticky ends” of the vector DNA comes into contact with the’ sticky ends of the selected gene; they splice together! • Now we have RECOMBINANT DNA • Recombinant DNA is a product of mixing the DNA of different organisms

Bacterial DNA • Single circular chromosome – only one copy = haploid – no nucleus • Other DNA = plasmids! bacteria chromosome plasmids

There’s more… • Plasmids – small extra circles of DNA – carry extra genes that bacteria can use – can be swapped between bacteria • bacterial sex!! • rapid evolution = antibiotic resistance – can be picked up from environment

How can plasmids help us? • A way to get genes into bacteria easily – insert new gene into plasmid – insert plasmid into bacteria = vector – bacteria now expresses new gene • bacteria make new protein gene from recombinant other organism cut DNA plasmid + vector glue DNA transformed bacteria

Grow bacteria…make more transformed gene from other organism recombinant plasmid + bacteria vector plasmid grow bacteria harvest (purify) protein

Splicing the gene we want into a Vector: Making and Delivering Recombinant DNA Cell with Normal Gene for human growth hormone Human Cell Sticky ends Bacterial Cell DNA recombination DNA insertion Bacterial chromosome Plasmid Using Bacteria as a Vector Bacterial cell for containing gene for human growth hormone

Figure 13 -10 Plant Cell Transformation Using a Recombinant Bacteria Plasmid to insert desired gen Into Plant Agrobacterium tumefaciens Gene to be transferred Cellular DNA Recombinant plasmid Inside plant cell, Agrobacterium inserts part of its DNA into host cell chromosome Plant cell colonies Transformed bacteria introduce plasmids into plant cells Complete plant is generated from transformed cell

Using a Virus as a Vector to insert Normal Hemoglobin Gene into Bone Marrow cells Gene Therapy Bone marrow cell Normal hemoglobin gene Chromosomes Genetically engineered virus : Nucleus Bone marrow Viruses Normally infect cells by injecting their DNA, So this process will occur naturally when viruses Containing Recombinant DNA contact cells

Applications of Genetic Engineering A. Transgenic Organisms: contain recombinant DNA 1. Transgenic Microorganisms: • Used as Vectors to carry desired gene to host • Used to Produce Human Hormones (Insulin, Human Growth Hormone) 2. Transgenic Animals: Contain DNA from another Organism (Recombinant DNA) • Genes to Make More Milk, Larger Eggs, More meat/less fat 3. Transgenic Plants: Contain Recombinant DNA • Disease Resistant • Larger Crop Yield B. Cloning : Using One source of DNA to make Exact copies • Reproducing new individuals from one parent’s DNA • Dolly

Dolly, the first Cloned Mammal A donor cell is taken from a sheep’s udder. Donor Nucleus These two cells are fused using an electric shock. Fused Cell Egg Cell An egg cell is taken from an adult female sheep. The nucleus of the egg cell is removed. The fused cell begins dividing normally. Embryo Cloned Lamb The embryo develops normally into a Foster lamb—Dolly Mother The embryo is placed in the uterus of a foster mother.

Flowchart Cloning A body cell is taken from a donor animal. An egg cell is taken from a donor animal. The nucleus is removed from the egg. The body cell and egg are fused by electric shock. The fused cell begins dividing, becoming an embryo. The embryo is implanted into the uterus of a foster mother. The embryo develops into a cloned animal.

Applications of biotechnology

I’m a very special pig! Got any Questions? 2006 -2007