Ch. 10 Genetic Engineering

•         Biotechnology - Application of biological systems (microorganisms) to obtain a product (food, antibiotics, vitamins)

•         Recombinant DNA technology – procedures by which a fragment of DNA (gene) of one organism is incorporated into the genom of a different organism

Goals of Genetic Engineering

•         Create organisms that synthesize products humans need (insulin)

•         Eliminate undesirable phenotypic traits (e.g. supression of ripening in tomatoes)

Tools and Techniques of Genetic Engineering

•         Restriction enzymes – major tool

•         Analysis of DNA – gel electrophoresis

•         Nucleic acid hybridization

•         DNA sequencing

•         Polymerase Chain Reaction - PCR

Restriction Enzymes

•         DNA cutting enzymes

•         They recognize and cut specific fragments of DNA  (sequences of nucleotides in DNA)

•         They leave single stranded sticky ends of DNA

•         DNA from different sources cut with the same restriction enzyme will produce the same type of sticky end

•         DNA is cut on a specific  Palindromic sequence

•         Palindromes are sequences that are identical when read in opposite directions in two strands

DNA Gel electrophoresis

•         Separation of DNA fragments based on their size

•         In agarose gel, DNA fragments are subjected to an electrical current

•         DNA molecule has a negative charge – moves toward the positive pole

•         Smaller fragments move faster

Nucleic Acid Hybridization

•         A fragment of a single-stranded nucleic acid (DNA, RNA) can hybridize (unite) with another fragment that has a complementary sequence of nucleotides

Hybridization with a probe

•         The method used to detect specific nucleotide sequence in an unknown sample by using a gene probe

•         Gene probe is a short segments of DNA of a known sequence

•         A probe carries a radioactive label 

DNA sequencing

•         A process in which exact sequence of nucleotides in a DNA segment is determined

Polymerase chain reaction - PCR

•         Technique by which small amount of specific DNA fragment can be amplified in vitro

•         What is needed?

–        PCR machine - thermal cycler

–        Target DNA that serves as a template

–        Supply of 4 nucleotides

–        DNA polymerase

–        Primers

•         Primers are short fragments of DNA that are complementary to the target DNA

One PCR cycle has 3 basic steps:

•         Denaturation: 94⁰C – separation of DNA strands

•         Priming: 50–65 ⁰C, primer attached to complementary strand of DNA

•         Extension: 72⁰C

     DNA polymerase extends the molecule by adding nucleotides

•         Typically we use 20-40 cycles – millions of copies of DNA

Recombinant DNA Technology – The procedure

•         A selected gene is removed from the genetic donor

•         This gene is incorporated into a vector (plasmid or virus)

•         The vector is inserted into the cloning host (bacteria, yeast)

Vectors

•         Vectors are DNA molecules into which a segment of foreign DNA can be incorporated (plasmids, transposons and viruses)

•         Vector’s characteristics:

–        self-replicating

–        circular shape

–        proper size – to be able to accept foreign DNA

–        must have a promoter

–        must have a gene for antibacterial resistance

Inserting foreign DNA into cells

Transformation

•         Plasmid from the surrounding environment is taken up by a cell

•         Cells have to be made competent - by soaking them in calcium chloride

Screening of bacteria that contain foreign DNA

•         The vector (plasmid) contains the gene for ampicilin resistance

•         Just those cells that have been transformed can grow on medium containing ampicilin

Synthetic DNA

•         DNA synthesis machine

•         Short fragments of DNA (120 nucleotides) can be synthesized

•         We must know the sequence of the DNA fragment that we want to synthesize

Applications of Recombinant DNA Technology

•         Pharmaceutical and Therapeutic Applications

–        Protein synthesis

–        Vaccines, DNA vaccines

–        Genetic screening

–        DNA fingerprinting

–        Gene therapy

Some examples

•         Hormone insulin, needed by diabetics

•         The gene for insulin was cloned into E. coli

•         Before it was obtained from the pancreases of slaughtered  animals

•         The first commercial success of recombinant DNA technology

•         Somatostatin - Human hormon used for treatment of giantism (an excessive secretion of growth hormone)

•         Before - 500,000 sheep brains were needed to produce 5 mg of somatostatin.

•         Today - 8 l culture of genetically engineered bacteria to obtain the equivalent amount

Subunit vaccines

•         A protein portion of the virus is cloned

•         Hepatitis B vaccine (Saccharomyces cerevisiae carries the virus gene on a plasmid)

•         Advantage - there is no chance of becoming infected during vaccination

DNA vaccines

•         A single gene from that pathogen is artificially copied and multiplied.

•         That gene is then injected into a muscle. Muscle cells tend to take up this gene and use it as one of their own genes, making the product

•         The immune system will recognize that product as foreign, and will start producing antibodies

•         A single gene from that pathogen is artificially copied and multiplied.

•         That gene is then injected into a muscle. Muscle cells tend to take up this gene and use it as one of their own genes, making the product

•         The immune system will recognize that product as foreign, and will start producing antibodies

Agricultural applications

•         Transgenic organisms – recombinant plants and animals altered by addition of genes from other organisms

•         Improving Crops

–        Herbicide resistance

–        Salt tolerance

–        Freeze resistance

–        Pest resistance

–        Improvements in nutritional value and yield

Creating transgenic plants

•         Agrobacterium tumefaciens - plant parasite that can incorporate its DNA into plant’s genom by using Ti plasmid

•         Ti plasmid can be engineered to contain a new gene

Some examples

Pest resistance - Tomato Plant with Bacillus thuringiensis toxin

Resistance to herbicides

•      Glyphosate (Roundup) kills all plants

•      Gene for resistance to glyphosate incorporated into   crop plants

•      Now farmers can kill weeds without killing crop

•         MacGregor tomatoes- Gene for pectin degradation suppressed, so they have a longer shelf life

Transgenic Animals

•         Why to create a transgenic animals?

•         The product (protein) can be collected in milk or semen

•         Many human genes have better expression in animals than in bacteria

•         Foreign genes are inserted into an embryo by using a virus or an injection

Gene Therapy

•         Mostly preliminary work

•         Missing or defective genes replaced with normal copies

•         Possible treatment for: cystic fibrosis, sickle cell anemia, some types of hemophilia, some types of diabetes

Antisense DNA and RNA

•         Antisense strand of DNA recognizes and binds to the complementary mRNA fragment

•         This results in blocking the expression (translation) of the harmful gene

•         Antisense drugs are being researched to treat cancers and other diseases

Genetic screening

•         Many genetic diseases can be detected by genetic engineering techniques

•         Technique: Southern blotting (Ed Southern 1975)

•         Inherited forms of breast cancer can be detected

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