GENETIC MODIFICATIONS SBI 4U: Metablic Processes GENETIC MODIFICATIONS Genetic engineering: altering the sequence of DNA Ideas established in early 70's by 2 American researchers, Stanley Cohen (worked with plasmids) and Herbert Boyer (restriction endonucleases) Initially had no commercial applications for their experiments, but things changed quickly. In 1976 Boyer cofounded Genetech, first biotech company to go public on the stock market. Section 1.3

SBI 4U: Metablic Processes 1978: somatostatin became the first human hormone produced by this technology Other examples: Insulin: over 90% diabetics are reliant on human insulin supplied by bacteria. Somatropin: used to treat human growth deficiency, from dwarfism, Turner's syndrome, also used for AIDS-associated wasting syndrome now Section 1.3

SBI 4U: Metablic Processes BIOTECHNOLOGY Biotechnology involves the manipulation of DNA and protein synthesis. Molecular biologists analyze and alter genes and their respective proteins Section 1.3

Examples Genetic screening: scanning for genetic mutations Gene therapy: the alteration of a genetic sequence in an organism to prevent or treat a genetic disorder by creating working proteins. Transgenic plants: inserting genes to provide new proteins, giving plants new properties DNA fingerprinting: analyzing pattern of bands that are unique to an individual. Human Genome Project...

SBI 4U: Metablic Processes Biotech Tools The tools the scientists use are very specific to DNA and its environment. The DNA first has to be cut out of the source organism The DNA has to be isolated DNA can then be introduced into host DNA Section 1.3

Recombinant DNA Recombinant DNA is DNA from one source organism being put into the DNA of a host organism.

SBI 4U: Metablic Processes 1) Cutting Out DNA Restriction Endonucleases / Enzymes are naturally occurring enzymes that act like a pair of molecular scissors to cut DNA in a predictable and precise manner, at a specific nucleotide sequence called a recognition site. Section 1.3

Discovery Hamilton Smith, John Hopkins University, won the Nobel Prize in 1978 for discovering restriction enzymes in bacteria. He found their main purpose was to cut foreign DNA that tried to invade a bacterial cell (ie DNA from a virus).

Naming System Restriction enzymes are named according to the bacteria from which they originate. BamHI is from Bacillus amyloliquefaciens, strain H. The I indicates it was the first endonuclease isolated from that strain. EcoRI -  from Escherichia coli BamHI - from Bacillus amyloliquefaciens HindIII - from Haemophilus influenzae (the one H. Smith found) PstI -  from Providencia stuartii Sau3AI - from Staphylococcus aureus AvaI -  from Anabaena variabilis

SBI 4U: Metablic Processes Recognition sites 4 – 8 base pairs in length. Palindromic: both strands have the same sequence when read in the 5' to 3' direction. Ex. HincII recognizes the following sequences: 5'-G T C GA C-3'      5'-G T T G A C-3'      5'-G T C A A C-3'      5'-G T T A A C-3' 3'-C A G C T G-5'   3'-C A A C T G-5'      3'-C A G T T G-5'     3'-C A A T T G-5' Section 1.3

The restriction enzyme EcoRI binds to 5'-GAATTC-3' 3'-CTTAAG-5' EcoRI breaks the phosphodiester bond between G and A, then it pulls apart the two strands by breaking the H-bonds between the complementary base pairs. Produces what are called sticky ends (unpaired nucleotides at each end).

SBI 4U: Metablic Processes Sticky vs. Blunt Other restriction enzymes like AluI produce blunt ends, or ends with no overhang. Sticky ends are usually more helpful to molecular biologists as they can easily be joined with other DNA fragments cut by the same restriction enzyme. Blunt ends are harder to fuse to a foreign DNA molecule. p281 #1-5 Section 1.3

SBI 4U: Metablic Processes A host must protect its own DNA from endonucleases. Methylases are enzymes that place a methyl group (CH3) on recognition sites This prevents the restriction enzyme from cleaving the DNA at that spot. Host DNA is methylated, but foreign DNA is not, so it can be cut by the host cell's restriction enzymes. Section 1.3

2)Isolating DNA Fragments SBI 4U: Metablic Processes 2)Isolating DNA Fragments Scientists make use of restriction endonucleases to cleave DNA into smaller fragments Gel electrophoresis is used to isolate the required gene segment from the rest of the DNA Section 1.3

Gel Electrophoresis The fragments of DNA will be run through a porous agarose gel using electricity. The fragments of DNA are pulled through pores in the gel due to their negative charge. Smaller fragments will move faster than larger because they can fit through the pores better.

SBI 4U: Metablic Processes http://www.stanford.edu/group/hopes/diagnsis/gentest/f_s02gelelect.gif Section 1.3

SBI 4U: Metablic Processes Steps: Solutions of fragments are placed in wells (depressions at one end of the gel) The DNA is mixed with a dye so it will be seen as it moves through the gel. Markers are usually put in the first well, These are pieces of DNA whose size is known. They help determine the length of the unknown DNA fragments. Section 1.3

SBI 4U: Metablic Processes The gel is submerged in a buffer solution and connected to a power source. The anode will be at the top and the cathode at the bottom. DNA is negatively charged, it will move away from the anode to the cathode. The power source is only left on for a set amount of time, so the fragments don’t move all to the end or run off the gel, you want them separated on the gel. Section 1.3

SBI 4U: Metablic Processes VIEWING THE GEL http://www.mcps.k12.md.us/departments/intern/stp/images/gel_electrophorsis.jpg http://www.life.uiuc.edu/molbio/geldigest/fullsize/geldraw.jpg The gel is stained with ethidium bromide which will cause the gel to fluoresce under UV light. The band of the DNA fragments can be seen and the researcher is able to compare samples from various sources or isolate a DNA fragment they want to purify. Section 1.3

3) INTRODUCING FOREIGN DNA INTO A HOST: PLASMIDS and TRANSFORMATION SBI 4U: Metablic Processes 3) INTRODUCING FOREIGN DNA INTO A HOST: PLASMIDS and TRANSFORMATION Plasmids are small (1000 to 200 000bp in length), circular DNA molecule independent of the bacterial chromosome. Plasmid DNA can be replicated using the bacterial cell’s machinery. http://www.rpgroup.caltech.edu/courses/PBL/images_dnascience/pZ%20Plasmid.gif Section 1.3

Plasmids Beneficial because they often contain important genes such as antibiotic resistance, heavy metal protection. Plasmids are used by biologists to incorporate genes they want replicated or transcribed/translated in vast amounts in little time into bacterial cells. Vector: vehicle used to introduce DNA into a host cell, ie a plasmid or virus.

3) INTRODUCING FOREIGN DNA INTO A HOST If we can cut genes out, we must be able to join them to foreign DNA. When sticky ends join together, DNA ligase recreates the phosphodiester bonds. Blunt ends cannot be joined by our own DNA ligase, they must be joined by , an enzyme from the T4 bacteriophage (virus).

SBI 4U: Metablic Processes STEPS: Restriction enzymes are used to cut out the gene from the original cell AND to open the bacterial plasmid. Once the foreign gene is isolated it can then be inserted into the plasmid. The plasmid is now considered recombinant DNA. http://employees.csbsju.edu/hjakubowski/classes/ch331/dna/plasmid.gif Section 1.3

SBI 4U: Metablic Processes TRANSFORMATION The recombinant DNA is then introduced into a bacterial cell. Sometimes a host cell must be manipulated to take up the foreign DNA plasmid. Section 1.3

Transformation: introduction of foreign DNA (usually by plasmid or virus) into a bacterial cell. Host cell: cell that has taken up foreign plasmid or virus and whose cellular machinery is being used to express the foreign DNA. Competent cell: cell that readily takes up foreign DNA.

4) Selection and Cloning SBI 4U: Metablic Processes 4) Selection and Cloning Cells that have been successfully transformed must be isolated (usually by antibiotic resistance) The vectors used for cloning usually carry an antibiotic-resistance gene. Growth of colonies on media containing the antibiotic indicates successful transformation. Section 1.3

Cloning Colonies are isolated from media and grown in culture to produce multiple copies (clones) of the recombinant DNA When the bacteria replicates the recombinant DNA plasmid, the new gene product will be formed multiple times (ie. the gene is cloned).

PCR – another means of copying DNA in large numbers SBI 4U: Metablic Processes PCR – another means of copying DNA in large numbers stands for Polymerase Chain Reaction, developed in the late 1980's by Kary Mullis; awarded Nobel Prize in Chemistry in 1992. Does not require a plasmid. The fragment is copied directly. Useful for forensic criminal investigations, medical diagnosis, genetic research. Only small amounts of DNA are needed. Section 1.3

SBI 4U: Metablic Processes PCR Process PCR is amplification of a DNA sequence by repeated cycles of strand separation and replication in the laboratory (DNA photocopying). After about 30 cycles more than 1 billion copies of the targeted area will exist (230). http://users.ugent.be/~avierstr/principles/pcrcopies.gif Section 1.3

Steps of PCR Strands are separated using heat DNA primers, synthesized in the lab, are created to complement the start of the target area to be copied. Temp is decreased and the primers anneal Taq polymerase (from bacteria) creates new strands of target area Sequence is repeated over and over on each of the new strands built

Restriction Fragment Length Polymorphism (RFLP) SBI 4U: Metablic Processes Restriction Fragment Length Polymorphism (RFLP) Entire genome is subjected to restriction enzyme digestion DNA run on an agarose gel, using gel electrophoresis Single stranded DNA transferred to a membrane http://homepage.smc.edu/HGP/images/rflp.gif Section 1.3

RFLP ssDNA hybridized with radioactive probes for specific regions (such as alleles or areas known as variable number tandem repeats, that lead to a specific disease). An X-ray film is developed, called an autoradiogram, and the pattern can then be used to identify a suspect, or detect a genetic mutation.

SBI 4U: Metablic Processes SEQUENCING DNA Sanger dideoxy method: uses DNA replication and dideoxy nucleotides to determine the complementary strand. Developed by Frederick Sanger and colleagues at Cambridge University in Great Britain in 1977. They used it to sequence the genome of a bacteriophage (viral DNA) 5386 base pairs long. Section 1.3

SBI 4U: Metablic Processes Sanger dideoxy method Dideoxy nucleotides are missing the -OH group on carbon 3 and therefore inhibit the process of replication. Every time one is added, the process stops and only small sequences are created. Section 1.3

These sequences can be run on a gel, and since they will run from shortest to longest, you can actually read the sequence by knowing which dideoxy nucleoside was used and therefore stopped replication at each point.

Fluorescent Detection of Oligonucleotides SBI 4U: Metablic Processes Fluorescent Detection of Oligonucleotides The Human Genome Project used a similar method, but also included fluorescence on each dideoxy nucleoside, so the A, G, T and C's lit up as different colours. Section 1.3

A computer reads the sequence from gel electrophoresis. Thousands of sequencers worked 24 hours a day, 7 days a week to decipher 3 billion base pairs.