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  • 1. Chemical Basis of Molecular Genetics molecular biology uses only a few key concepts for all of the work fit ideas together, and technique are easier to understand A. complimentary sequences bind to each other 2 strands of nucleic acids WANT to stick together (ie. sticky ends) B. enzymes that work in cells work in test tubes too DNA polymerase synthesizes DNA from primers DNA ligase ties 2 pieces of DNA together restriction enzymes cut DNA at particular bases recombinases/transposases work on natural and manipulated DNA C. labeling one thing allows anything bound to it to be identified D. separation by size using electrophoresis E. transfection of DNA into other cells
  • 2. Restriction Endonucleases Restriction endonucleases : enzymes which cleave within sequences of DNA at particular sequences usually cleave palindromic sequences palindrome : sequence that reads the same way in both directions ie. complementary sequence is identical to the other side GGGCCC restriction site for Sma1 (blunt) CCCGGG restriction site for Xma1 (sticky) most restriction enzymes are protein homodimers, forcing them to cut palindromic sequences restriction fragment : piece of DNA cut by restriction enzymes at both ends
  • 3. sticky end -- cleaved piece of DNA with a short overhang of single stranded DNA (that can be used for sequence specific DNA ligations) G GGCCC note the single stranded DNA is complimentary CCCGG G blunt end -- cleaved pieces of DNA without overhanging nucleotides (any 2 cut pieces can be joined together using T4 or 7 DNA ligase) GGG CCC CCC GGG Restriction Endonucleases
  • 4. Restriction Endonucleases restriction enzymes generate fragments of different sizes from DNA mutations or polymorphisms within the DNA sequence can change the size of a fragment fragments can be cut out of an agarose gel to purify them from other pieces there are many different restriction enzymes, many with different sequences of DNA that are cut (most commonly 6 base palindromes) restriction map : pattern of restriction sites in a given DNA sequence size polymorphisms 6kb 5kb 4kb 3kb 2kb
  • 5. DNA hybridization agarose gel electrophoresis separates DNA pieces by their size small pieces migrate faster than large ones
  • 6. DNA hybridization denaturation : separation of 2 strands of DNA renaturation : coming together of 2 strands of DNA (not necessarily the same 2 strands that were denatured!) DNA needs to be transferred to a nylon filter (positively charged) from an agarose gel, usually done by blotting (either wicking action or electrophoretically) probe : piece of nucleic acid labelled so it can be detected later hybridization : incubating a probe with a filter bound with DNA to identify a piece of complimentary DNA all DNA labeled DNA
  • 7. DNA hybridization 1a) Separate nucleic acids 2a) transfer to a nylon filter 3a) denature DNA on filter 4a) block filter so probe can't bind 1b) obtain a nucleic acid probe 2b) label nucleic acid ( 32 P, biotin, digoxigenin, fluorescein, etc) 3b) make sure probe is denatured 5ab) mix labeled probe + nonspecific DNA with denatured DNA on filter 6ab) wash excess probe away using (often) temperature to control binding low stringency : allow probe with related sequences to stay bound high stringency : only allow very close matched sequences to stay bound 7ab) detect labeled probe-- identifies specific sized pieces of DNA
  • 8. Southern blot : pieces of DNA separated on a gel after restriction digest and probing using some labeled nucleic acid (usually DNA) Northern blot : RNAs are separated on a gel and hybridized with a probe DNA hybridization
  • 9. DNA hybridization
  • 10. cloning : taking a piece of DNA and ligating it into a self-replicating piece of DNA (usually a plasmid or bacteriophage) plasmid : independent DNA circle that replicates separately from rest usually present in multiple copies per cell different plasmids can replicate in bacteria or eukaryotes or both modern plasmids contain many restriction sites that can be used to clone in many different sequences for various purposes different plasmids are for DNA sequencing, protein overexpression, transgenic organisms, eukaryotic protein expression even with restriction digesting and hybridization, it is difficult to clone the specific genomic DNA that you're interested in Cloning: Making Many Copies of One DNA
  • 11. Reverse Transcriptase RNA viruses use RNA as their primary genetic material-- ie. HIV these viruses have a very special enzyme called reverse transcriptase which can make a DNA copy from the RNA Molecular biologists use reverse transcriptase to make 'copy' DNA or cDNA (DNA made from mRNA) allows scientists to study exactly what mRNA (i.e. proteins) are made reverse transcriptase binds DNA, makes 1 strand many times over primer + DNA polymerase makes double stranded DNA
  • 12. cDNA cDNA is a tool that is commonly used by molecular biologists ie. isolate messenger RNA using its polyA tail-- specific for mRNA make cDNA from that mRNA using reverse transcriptase ligate cDNA into a vector (usually a bacterial plasmid or bacteriophage) put that new recombinant DNA into bacteria by transfection now you have foreign, maybe human, DNA in the bacteria for screening by hybridization library : collection of foreign DNA cloned into a vector for isolation and analysis many different sizes and sequences
  • 13. Polymerase Chain Reaction: PCR relies on several key features to work: 1) small pieces of DNA can hybridize at moderately high temperatures 2) must have the DNA sequence to design synthetic primers 3) use a DNA polymerase that withstand high temperatures which are able to denature the DNA strands first identified from Thermus aquaticus , a bacteria found in thermal pools now at least half a dozen thermostable polymerases for sale PCR amplification is exponential-- newly synthesized strand can be used as a template for the next cycle of reactions oligonucleotides (short DNA pieces) can be chemically synthesized
  • 14. Polymerase Chain Reaction: PCR separate and hybridize extend from the primer separate and hybridize extend from the primer many primers and polymerases around 2nd round, 4 oligos ready to go all new strands will be templates next cycle- exponential
  • 15. How to clone your DNA figure out what DNA you are interested in: genome sequences, related species sequences, messenger RNA from a particular tissue obtain DNA for what you want (reverse transcribed RNA, PCR primers, etc.) make PCR primers with desired restriction sites at both ends and amplify desired DNA coding for what you want (usually a protein) note the new longer size!
  • 16. How to clone your DNA restriction enzyme sites at the ends of the PCR primers make cloning much easier-- allows fast, accurate movement to another piece of DNA find a restriction seqeunce that is NOT in the piece of DNA you are cloning-- otherwise you will cut your PCR band in the middle as well choose your restriction site based on what vector you are cloning into vector : another DNA molecule which can accept a piece of DNA; a carrier for your piece with specific properties sticky ends (asymmetric cutting) are better than blunt-- ligation is more efficient, uses short overlap for specificity EcoR1 EcoR1 cut with EcoR1 and run an agarose gel uncut cut
  • 17. How to clone your DNA plasmid : particular type of circular DNA vector most commonly used in DNA cloning experiments usually 3-6 kb long will usually have at least 1 multi-cloning site : region of the plasmid that has been engineered to include a lot of different unique restriction sites this gives lots of opportunities to clone your DNA into the vector
  • 18. Plasmid maps plasmids have several requirements that allow them to be used bacterial origin of replication eukaryotic origin of replication polyadenylation sequence multicloning site eukaryotic promoter prokaryotic/ eukaryotic promoter antibiotic resistance gene (kanamycin/ neomycin)
  • 19. How to clone your DNA cut plasmid and insert (what you are cloning) with 1 or 2 restriction enzymes gel purify your cut DNA on an agarose gel ie. run a gel, then cut your band out of it mix your cut vector with insert and DNA ligase DNA ligase : enzyme which joins 2 fragments of DNA-- sticky ends are more efficient T4 DNA ligase : viral enzyme that can stick blunt ends of DNA together
  • 20. Recombinant DNA with DNA ligase, it's possible to join any 2 pieces of DNA together for example, if you want to make an entirely new protein that is a hybrid or chimera of two other proteins, you design your two PCR pieces, ligate them together into a vector and off you go ie; there are a group of proteins isolated from jellyfish that fluoresce it's possible to fuse a protein of interest to a naturally fluorescent protein and watch what happens to that protein in a living cell ie. can you change where the protien is localized
  • 21. Transfection transfection : process of putting foreign DNA into another cell three common ways of doing this 1) electroporation : using an electric field to shock cells into taking up the foreign DNA-- extremely efficient 2) heat shock : raising the temperature of specially treated bacteria so that they take up the foreign DNA 3) lipofection : using a mixture of lipids bound to DNA that will fuse with the cell membrane, releasing DNA inside cell
  • 22. More DNA Vectors cosmids are similar to plasmids with circular DNA but have some bacteriophage sequences which allow them to go up to about 50 kb bacteriophage : virus that infects bacteria  phage are the most commonly used-- usually for making libraries very useful because they can be purified to high titre (particles/mL ) lentivirus : RNA virus similar to AIDS manipulated to be unable to reproduce AND to infect almost any cell commonly tested for in vivo uses artificial chromosomes : several varieties, including yeast and bacterial basically very large recombinant DNAs-- 300 kb not uncommon in yeast, have centromeres, rep. origins, etc like any other chromosome
  • 23. 1) Design PCR primers (with restriction sites) that amplify your DNA use restriction sites that will not cut in your plasmid or DNA! 3) cut the plasmid and the PCR fragment with restriction enzymes 4) separate the cut pieces on an agarose gel-- cut out appropriate bands 5) mix pieces and ligate together using DNA ligase 6) transfect the ligation reaction into bacteria select for transfected cells using antibiotic resistance 7) grow up the bacteria and purify your new plasmid 8) make sure the plasmid is correct by DNA sequencing 8 Step Program for Cloning DNA
  • 24. dideoxy DNA Sequencing polymerases use a free 3' hydroxyl to add a new dNTP to a growing DNA chain, releasing 2 phosphates dideoxynucleotides end DNA synthesis no 3' hydroxyl for the next dNTP to bind often run 4 reactions, one each with ddA, ddC, ddG, and ddT to stop randomly at each base label each ddNTP with 35 S or fluorescence
  • 25. combining dideoxy nucleotides with PCR, can sequence small amounts of DNA-- linear amplification of pieces instead of exponential newer method- use different fluorescent colors representing the different base; can have all 4 ddNTPs in one lane- gives more sequences per gel, with 1 sequence per lane dideoxy DNA Sequencing
  • 26. dideoxy DNA Nucleotides Bacterial and viral polymerases are somewhat different than eukaryotes some viruses use RNA as their genetic material-- note that this requires an enzyme that can recognize RNA as a genetic template reverse transcriptase : enzyme that synthesizes DNA from RNA template because it is a polymerase, it is required in the viral life cycle
  • 27. dideoxy DNA Nucleotides some dideoxynucleotides have fairly high affinity for the polymerase ie. AZT, DDI are nucleotide analogs that block RNA polymerases (AIDS) analog : molecule that is related to the real one, but can't undergo a particular reaction; ie. transition state analogs dideoxynucleotides are common drugs to fight AIDS- by blocking reverse transcriptases preferentially, viruses can't replicate and cells can
  • 28. Genomic Sequencing Fragment the human genome using X rays-- clone large fragments into artificial chromosomes order the artificial chromosomes and subclone into cosmids order cosmids along the artificial chromosomes sequence DNA from cosmids in a random fashion-- shotgun sequencing put lots of overlapping fragments into a computer to sequence a cosmid use overlapping cosmid sequences to map artificial chromosomes use overlapping artificial chromosomes to generate a complete sequence build up overlapping short sequences
  • 29. Reverse Genetics Mendel started with his mutant phenotypes to come up with his rules Today, we know DNA sequences of many genes but not their phenotype reverse genetics : generation of an organism expressing your desired gene easiest way-- engineer a retrovirus to express your gene via cloning infect embryonic stem cells with your retrovirus-- integrates randomly into genome as part of the retroviral life cycle, then inject into embryos
  • 30. Reverse Genetics- Transgenics
  • 31. Reverse Genetics gene targeting : procedure for introducing a specific mutation into 1 gene in the organism first, obtain the genomic DNA sequence of your gene of interest next engineer a large plasmid with your desired mutation in that genomic DNA (stop codons, deletions, insertions, entire replacements, etc)
  • 32. Reverse Genetics transfect embryonic stem cells with your plasmid and look for ES cells that have undergone recombination (usually using PCR or hybridization looking for restriction fragment length polymorphisms) using antibiotic resistance inject your mutant ES cells into blastocysts as with retroviruses
  • 33. Reverse Genetics rescue : ability of a cloned fragment of DNA to recover a wild type phenotype in the organism ie. putting back a deleted gene to recover full function transformation rescue : use of genomic DNA sequences to reproduce the expression pattern of the wild type gene regulatory promoter and enhancer regions are difficult to identify-- generally relatively small and spread out compared to bacteria minimal regulatory region : the smallest amount of DNA to reproduce the normal pattern mRNA regulatory region (enhancers and silencers)
  • 34. Applications of Genetic Engineering growth hormone is a small protein that can increase the size of an animal you can make a transgenic animal overexpressing growth hormone can also inject human growth hormone made in bacteria into humans as a treatment for dwarfism insulin for treating diabetes is another protein that can be made in bacteria
  • 35. Applications of Genetic Engineering plants can also be engineered -- GM (genetically modified) crops other strains of plants have been generated to be resistant to a particular herbicide-- now you can spray a field with that herbicide and not kill your crops while killing weeds other plants express a natural pesticide-- kills insects that try to eat the plants What do these new organisms mean for human consumption/health?
  • 36. rice has been designed to make  carotene using 4 genes from 2 different organisms-- daffodils and bacteria 400 million people are deficient in  carotene (Vitamin A) 'ice-minus' bacteria-- live symbiotically with the plants and provide an 'anti-freeze' protein to protect the plants from frost Applications of Genetic Engineering drought resistant rice survives much better-- may increase yields 20% rice is a major choice for engineering because so many people eat it
  • 37. Applications of Genetic Engineering gene therapy : use of recombinant DNA (often viruses) to correct genetic defects has been used successfully to treat severe combined immunodeficiency syndrome (SCID) works best in stem cells that will give rise to well understood progeny harder with things like neurons-- must become part of an existing circuit being tested with Parkinsons, Alzheimer's, stroke patients
  • 38. Functional Genomics functional genomics : expression pattern of all the genes in all the tissues of the body and all the normal and disease states ie. changes in gene expression during development, aging, cancer, etc only possible with the sequencing of the human genome~ 25,000 genes northern blotting was the hybridization technique of separating all the mRNA on a gel, blotting it, and using a probe to see a single mRNA microarrays reverse the blotting process-- take all 25,000 genes and put them in 25,000 different spots (each very small) according to a pattern (do it in at least duplicate so you have reproducibility) label mRNA from 2 different tissues using 2 different fluorescent dyes hybridize labelled mRNA to the array; wash; analyze amount of color
  • 39. Functional Genomics green = mRNA #1 red = mRNA #2 yellow = overlap of red and green intensities of each color represent the amount of that mRNA in the inital samples each spot represents a different mRNA this picture represents 800 northern blots!!!