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Final Review - Roy Lectures

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McGill University
Biology (Sci)
BIOL 200
Mathieu Roy

L17. MOLECULAR GENETICS METHODOLOGIES BACTERIAL RESTRICTION ENZYMES CUT DNA AT SPECIFIC SITES DNA can be amplified from this vector vector: plasmids (exist endogenously in bacteria, or they can be manufactured) o polylinker - where the magic happens sites that can be recognized by endonucleases o endonucleases: cut DNA at specific palindromic 6 sequences that occur relatively infrequently in the gene genomic DNA can be inserted into the vector (after it is digested by the specific endonucleases) Q. what is the use of having multiple polylinkers in the vector? o 1 gene => 2 differential enzymes => facilitate directional cloning insert the gene in the correct orientation o 2> genes => use multiple restriction enzymes able to clone multiple genes, and insert them in the correct orientation STUDYING GENES OF INTEREST: DNA LIBRARIES 1. digest entire genomes a. first: mechanically b. second: restriction enzyme digests 2. sequences are kept in genomic libraries o one genome fragment per plasmid cDNA libraries - represent mRNA o tissue specificity, abundance genomic libraries - copies of the DNA present in the genomic/chromosomal context o intergenic regions, introns, exons, repetitive sequences mRNA CAN BE CONVERTED TO cDNA USING RETROVIRAL REVERSE TRANSCRIPTASE (RT) if you want to take a snapshot of a transcription (all mRNA present within a cell) reverse transcription => make a cDNA library from mRNA in vitro any DNA synthesis requires a primer o RNA synthesis does not require a primer 1. grin up all mRNA 2. hybridize with poly-t primer 3. remove RNA by RNA-alkali or RNaseH 4. synthesize a complementary DNA strand using polymerase mRNA CAN BE CONVERTED TO cDNA: USING REVERSE TRANSCRIPTASE (RT) 1. prime poly-A tail with a single stranded poly-T nucleotide 2. RT uses the poly-T nucleotide => initiates single strand DNA synthesis complementary to mRNA template 3. RNA removed 4. poly dG adapter: is annealed to the 3' end of the DNA o covalently linked by a phosphodiester bond 5. poly dC primer: initiates synthesis of the second DNA strand o poly dC is complementary to poly dG 6. DNA polymerase I progresses through the remaining hybrid regions => extends the second strand alternative: leave a little bit of RNA at the 5' end to use as a primer TRANSFORMATION - adding vectors to prokaryotes (bacteria) TRANSFECTION - inserting vectors to eukaryotic cells SPECIALIZED VECTORS PERMIT EFFICIENT EXPRESSION IN HIGHER EUKARYOTIC CELLS a. TRANSIENT TRANSFECTION o transfect cultured cells with a vector protein is expressed from cDNA in plasmid DNA o not all cells will take up the plasma o eventually all cells will get rid of the plasmid no centromere (no faithful segregation) => loose plasmid b. STABLE TRANSFECTION (do not use the term transformation) o vector contains a antibiotic resistance gene; therefore only cells which contain the vector will survive whenever cells lose the vector they die o when the vector is transfected into the host chromosome => becomes stable greater percentage of integrants if the plasmid gene is homologous to the vector gene o final product: all cells will express the transgene MOLECULAR PROBES CAN BE USED TO FIND A NEEDLE IN A HAYSTACK if you run a complex mixture on a gel => you need a way of identifying the target probes - specific for target recognition (radioactively labeled) o nucleic acid probe - complementary sequence o protein probe - antibodies 1. bind mixture to a solid support - nitrocellulose or nylon 2. probe used to identify target 3. wash off non target stuff 4. target detection RADIOLABELED SINGLE STRANDED OLIGONUCLEOTIDES if you know the sequence of the protein, but want to detect the mRNA which corresponds to the protein o known: a.a. sequence o unknown: nucleotide sequence make a probe for all codon possibilities, for each a.a. => creates a degenerate pool (due to wobble pairs) POLYNUCLEOTIDE KINASE - protein which phosphorylates macromolecules o phosphorylate either the 5' or 3' end of the macromolecule o PNK binds: target & ATP => transfers the gamma phosphate (ATP) to the free hydroxyl at the 5' end of the oligonucleotide gamma phosphate must be radioactive PCR CAN BE USED TO MAKE RADIOLABELLED DNA PROBES 1. denature DNA 2. anneal primers 3. elongation of primers with dNTP monomers o deoxyribo-nucleotides: contain a radioactive alpha phosphorous (most proximal to the sugar and base) if beta or gamma are radioactive then you lose the radioactivity since these are discarded o radioactive label each dNTP or pick one dNTP probes must be denature prior to use ANALYSIS OF NUCLEIC ACIDS BY TRANSFERRING TO SOLID STATE SUPPORTS (NYLON, NITROCELLULOSE) charged molecules are pushed towards the electrode of the opposite charge larger molecules are retarded due to the pores in the gel once the charge is stopped, the macromolecules will begin to disperse in a Brownian manor o => transfer to a solid support where they will maintain their position solid support is put ontop of the electrophoresis o macromolecules are pushed up to the solid support state o maintain position for (~1 month) normally you cannot use probes on the gel o much easier to use probes on the solid state DNA: nylon, nitrocellulose o southern RNA: nitrocellulose o northern proteins: o western o must be denature by SDS before all substances must be denatured before they are analyzed o mRNA => denature before electrophoresis because of secondary structure ONCE COVALENTLY BOUND THE LEVELS AND POSITIONS ARE PERMANENTLY RECORDED AUTORADIOGRAM: nitrocellulose hybridized with labeled DNA or RNA probe o film of the nitrocellulose NUCLEIC ACID HYBRIDIZATION TECHNIQUES AND DNA DETECTION alleles - same gene location but different locus (ex 200bp) in one allele there is not restriction cut site, in the other allele there are no restriction cut sites o can analyze on a gel to see if the person has one or two fragments o heterozygote: shows both 1. amplify the whole locus 2. digest with restriction endonucleases 3. probe for single-copy region PCR CAN BE USED FOR GENOTYPING ex. BRCA1 (breast cancer related 1) gene can be detected o ex.risky allele can be cut homozygous for risk allel: 2 fragments at the bottom of the page homozygous for non-risk allele: 1 fragment at the top of the page oligonucleotides made at least 20NT long o so that there is ex low probability of not seeing an enzyme single nucleotide polymorphism: have to know which allele it is abolished in NORTHER ANALYSIS: RNA Q. what is a possible explanation of different levels of mRNA in a particular organ? o RNA editing o alternative spicing 3 EUKARYOTIC RNA POLYMERASES HAVE SPECIALIZED TASKS** (memorize slide) RNA POLYMERASE II
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