Recombinant DNA•recombinant DNA – techniques in which genes from two different sources - often different species - are combined in vitro into the same molecule•This works because the genetic code is universal•genetic engineering – the direct manipulation of genes for practical purposes•DNA technology has resulted in biotechnology, the manipulation of organisms or their components to make useful products•DNA technology is now applied in areas ranging from agriculture to criminal law

• genetic engineering is possible because of restriction enzymes (restriction endonucleases):

• Very specific – recognize and then cut DNA molecules at specific base sequences called a restriction site (recognition sequence)– These are often a symmetrical series of four to

eight bases on both strands running in opposite directions.• If the restriction site on one strand is

3’-CTTAAG-5’, the complementary strand is 5’-GAATTC-3’.

• In nature, bacteria use restriction enzymes for protection to cut foreign DNA (from invading viruses)

• Restriction enzymes cut the covalent bonds of both strands, often in a staggered way creating single-stranded sticky ends.– Sticky ends will form

hydrogen-bonded base pairs with complementary sticky ends on other DNA molecules cut with the same restriction enzyme.

• DNA ligase bonds the complementary sticky ends together

• Restriction enzymes and DNA ligase are used to “cut and paste” DNA pieces together

Bacterial Transformation•Bacterial Transformation – scientists put new genes into bacteria to develop organisms that are beneficial to people – uses include:

–Bacteria that can produce hormones such as human growth hormone and insulin

–Bacteria that eat oil slicks

Escherichia coli• Often used for genetic engineering• Common inhabitant of human colon – easy to

get• Can be easily grown in suspension culture in a

nutrient such as Luria broth• Has a simple circular chromosome with about

1/600th the haploid amount of DNA in a human cell

• E. coli often contain small circular DNA molecules called plasmids (extrachromosomal) – confer a particular trait such as resistance to antibiotics– So we can easily introduce our own plasmids to

produce desired products

– Plasmids are produced by cutting desired DNA (using restriction enzymes) and inserting a gene into a plasmid to act as a carrier

– The gene is often inserted into a plasmid with genes for antibiotic resistance so that the transformed bacteria can be easily selected from other cells that did not pick up the plasmid

• In nature, genes can be transferred between bacteria in three ways:– Conjugation – mating process during which

genetic material is transferred from one bacterium to another of a different mating type

– Transduction – a virus acts as a vector (carrier) to transfer small pieces of DNA from one bacterium to another

– Bacterial Transformation – involves the transfer of genetic information into a cell by direct uptake of the DNA (occurs only rarely in nature)

Transformation in the Laboratory

• Transformation was first performed in the laboratory by Griffith and later by Avery, MacLeod, and McCarty (experiment using mice and pneumococcus bacteria)

• Bacteria can take up DNA only during the period a the end of logarithmic growth – cells are said to be competent (can accept DNA that is introduced from another source)

• E. coli competence can be induced under carefully controlled chemical growth conditions

• Plasmids can transfer genes and act as carriers for introducing DNA from other bacteria or from eukaryotic cells

• E. coli cell membrane is weakened using ice cold CaCL2

• E. coli cells are then “heat shocked” to induce them to take up the plasmid

• Sterile technique must be used• Transformation Lab – we will transform bacteria by

introducing a plasmid that will convey resistance to the antibiotic, ampicillin

• Ampicillin kills bacteria by interfering with their ability to make cell walls

DNA Profiling

• Restriction Enzymes are also used for DNA profiling– Creating a pattern of DNA bands on a gel

• Because the restriction site (recognition sequence) usually occurs (by chance) many times on a long DNA molecule, a restriction enzyme will make many cuts

• Result: production of fragments of DNA of various lengths – Restriction Fragment Length Polymorphs (RFLPs)

• Since all individuals have unique sequences of DNA, restriction enzymes cut each individual’s DNA into different sized RFLPs

• The RFLPs are then separated by gel electrophoresis resulting in a bar-like pattern

• Electrophoresis means “to carry with an electric current”

• Different sized RFLPs will be carried different distances by an electric current as they migrate through an agarose gel inside a gel box – Electricity is run through the gel box creating a

positive end and a negative end• Negatively charged DNA migrates from the

negative end of the gel box through the pores in the gel to the positive end of the gel box

• Smaller RFLPs will migrate farther than larger pieces, spreading the RFLPs across the gel in a bar-like pattern

• Stain is used to make the DNA bands visible

SEM photo of a 1% LE Agarose gel at 22kX magnification

Uses for DNA profiling

• Allows scientists to compare DNA from various organisms and identify a particular individual (DNA can be extracted from blood, saliva, hair roots, and skin)

• Crimework: rape and murder cases (forensics)

• Paternity suits• Missing persons and unidentified bodies• Immigration disputes• Animal work - breeding

Agricultural uses of DNA technology Animal Husbandry – many farm animals are

treated with products made by recombinant DNA methods (examples include vaccines, antibodies, and growth hormones)

– some milk cows are injected with bovine growth hormone (BGH) made by E. coli, in order to raise milk production

– BGH also improves weight gain in beef cattle

Transgenic animals – animals that contain genes from another species have been developed for agricultural use (examples include beef and dairy cattle, hogs, sheep and several species of commercially raised fishes)– modified DNA can be introduced into diary cows so that

they produce human proteins – protein is produced in the milk – examples of medically important proteins that have been produced in transgenic mammals include:

• blood clotting Factor VIII to treat hemophilia• alpha-1- antitrypsin which helps protect the lungs

from damage during infections– rainbow trout and salmon that are given a foreign

growth hormone can reach in one year a size that usually requires 2 to 3 years of growth

Genetic engineering in plants

– plants have been genetically altered to receive herbicide resistance (several strains of cotton) – allows them to be resistant to herbicides used to kill weeds

– some crop plants are being engineered to resist infectious pathogens and pest insects – reduces need to apply chemical insecticides

• first genetically engineered fruits approved by the FDA for human consumption were tomatoes engineered with antisense genes that retard spoilage– researchers isolated gene responsible for

ripening– they prepared a gene who's template strand had

a base sequence complementary to the normal gene – an antisense version of the gene

– when spliced into the DNA of a tomato plant, the antisense gene is transcribed into RNA that is complementary to the ripening gene’s mRNA

– the antisense RNA binds to the normal mRNA, blocking the synthesis of the enzyme causing ripening and spoilage

Benefits and Possible Harmful Effects of Genetic Modification

• See handouts and Clegg pg. 127-128

Cloning• Clone – a group of cells, organisms, or

genes that are exact copies of each other– Gene cloning – replication of donor genes in

bacterial or other host cells– donor gene inserted into a bacterium is copied

every time the plasmid containing it replicates – genes can be cloned by growing genetically engineered bacteria

Polymerase Chain Reaction• cloning a gene through genetic engineering can be time-

consuming and requires an adequate DNA sample as starting material

• PCR technique allows researchers to amplify a tiny sample of DNA millions of times in a few hours

• DNA polymerase uses nucleotides and primers to replicate a DNA sequence in vitro, thereby producing two molecules

• Two strands of each molecule are then separated by heating and replicated again, so then there are four, double-stranded molecules

• After the next cycle of heating and replication there are eight molecules, and so on

• Number of molecules doubles with each cycle• PCR is useful in amplifying tiny samples of DNA ranging

from crime scenes to archaeological remains

• Cloning organisms – cloning sometimes occurs naturally (twins, asexual reproduction)

• organisms can be cloned artificially (sheep, rabbits, toads and other sexually reproducing animals have been cloned by dividing up an embryo and transplanting them into surrogate mothers)

Cloning of Dolly1.sheep cloned from a non-reproductive cell2. cell taken from udder of donor adult and cultured

in lab for 6 days3.unfertilized egg taken from another sheep –

nucleus removed4.egg without nucleus is fused with donor cell using

a spark of electricity5.embryo resulting from fusion of udder cell and egg

transferred into the uterus of a third sheep who acts as the surrogate mother

6.surrogate mother gives birth to lamb – lamb is genetically identical to sheep that donated udder cell

Stem Cells• Cells that retain their ability to divide and

differentiate into various cell types

• Plants contain stem cells in their meristems (reason why a cutting can grow into a new plant)

• Embryonic stem cells are pluripotent – can form any type of cell in an organism or can form a complete organism

• Adult stem cells can divide to form new body tissue cells (i.e. blood stem cells)

Therapeutic Uses of Stem Cells• Embryonic stem cells are the most flexible

and can grow into any type of mature cell– Parkinson’s and Alzheimer’s Disease can be

potentially treated by implanting stem cells that could replace the damaged cells

Ethical Issues surrounding therapeutic cloning

• Therapeutic Cloning is the creation of an embryo to supply embryonic stem cells for medical use

– Raises issue of whether it is right or wrong to generate a new human embryo for medical research

• Two distinct forms of cloning:1. Reproductive cloning – making copies of entire

organism2. Therapeutic cloning – making copies of embryonic

stem cells• Opinions vary about whether both forms are

right/wrong or if one or the other is acceptable

Human Genome Project• A project that involved mapping the entire human

genome – determined the order of all the bases in human DNA

• Outcomes of the HGP:

1. Determine how many individual genes we have and how they work.

2. Locating and determining the cause of genetic disorders.

3. Development of gene therapies to treat genetic disorders.

4. Comparing genetic makeup of human populations to determine ancestries and how humans have migrated and mixed their genes with other populations over time.