Biol 200 – Professor Richard Roy Lecture 17 – Molecular Genetics Methods II E. Coli and bacteriophage lambda are the workhorses of molecular biology Most higher organisms use similar processes as bacteria and viruses (phage) as to replicate, transcribe, and translate their essential cellular molecules Purified components (i.e. enzymes) facilitated the rapid progress Paul Berg and others were the first to successfully introduce exogenous DNA fragments into bacteria to produce a recombinant gene product This milestone ushered in the era of recombinant DNA technology Cloning – Easy to Misunderstand Clone: o Group of organisms produced from one stock or ancestor o One such organism o Person or thing regarded as identical to another It can be used in different circumstances to point out different procedures o Early frog embryo . Clone by physically splitting . Two frogs develop from same zygote . One single blastomere can develop more than one o Cloning by: replacing the nucleus of an egg cell by the nucleus of an adult, somatic cell, implanting a resulting embryo . Get rid of the nucleus in an oocyte and take a nucleus from a fully differentiated cell, e.g. cell from mammory tissue . Put nucleus in the oocyte and reprograms the cell . E.g. cloning of Dolly . Can give rise to totipotent cells through this reprogramming mechanism o There are certain mutants that have been identified with strange morphological changes . E.g. drosophila head with legs growing where the antenna should be – antennapedia . Geneticists can identify the messed up chromosome . They take chunks of chromosomes and chew it down in more simple bits of DNA that you can work with . Find chromosomes in a DNA context and find a way to make tons of that DNA . Can put DNA in plasmids and amplify o Need lots of material to understand sequences . We use plasmids that can replicate autonomously to produce large quantities . Can carry out analyses on large quantity Bacterial restriction enzymes cut DNA at specific sites EcoRI (and other enzymes like it) cuts DNA at a specific six nucleotide, palindromic sequence (GAATTC) that occurs relatively infrequently throughout the genome The reaction bi-products have overhanging ends that are fully complementary and are thus “cohesive” Specialized plasmids called vectors were engineered to carry out specific functions Polylinkers (or multiple cloning sites) facilitate the introduction of DNA fragments Differential digestion facilitates directional insertion of desired DNA at specific sites Remember that vectors can close back on itself because the cohesive ends can stick back together If use only one enzyme, it can go in either direction Digestion of a vector with two different enzymes facilitates directional cloning o Asymmetry in the digestion o Plasmid can’t close up on itself because the ends are different o DNA interested in can only go in one way – EcoRI will only interact with EcoRI ends and SphI will only interact with SphI ends Studying Genes of Interest: DNA Libraries Permanent collections of genes can be obtained and maintained in DNA libraries o Purify DNA or chromosome of an organism o Cut that DNA or chromosome with a restriction enzyme digest o Take the whole mixture and clone it into vectors so that every little piece of the chromosomes will be incorporated into those particular vectors o Anneal them using DNA ligase o Maintain all the plasmids as a library o Should have all DNA represented in a library that you can grow and maintain o If you want to identify a chromosome or specific DNA sequence, you can take library anytime and fish for the particular plasmid that has that insert that corresponds to the DNA of interest Genomic libraries contain copies of the DNA present in genomic/chromosomal context (intergenic regions, introns, exons, repetitive sequence) cDNA libraries represent mRNA (tissue specificity, abundance) mRNA can be enriched from a starting population of total RNA (tRNA, rRNA) You want to purify the mRNA from a given source o It has all the qualities that allow us to enrich it away from the more abundant ribosomal RNA and tRNAs that exist in the cell The poly-A tail of mRNAs enables their purification via oligo-T-nucleotides linked to a solid support Can use a column with oligo-T to run RNA with a poly-A tail which will bind to the oligo-T nucleotides in the column while everything else goes through Can then elute the column and the elution mixture will contain a great enrichment of mRNA – all because of poly-A tail mRNA in that tube should be representative of all the mRNAs that were formed in a cell at a given time But they degrade very rapidly – must make it into cDNA Using the oligo-T paired to poly-A tails as primer, reverse transcriptase (RT) is used to synthesize the complementary or cDNA o RT is a viral enzyme that allows viruses to use RNA as a template to make DNA o Can make dsDNA that can be maintained (cDNA is single-stranded and can act as a template) mRNA can be converted to cDNA using retroviral Reverse Transcriptase (RT) If you make the first strand of DNA from an mRNA template, you can make a double-stranded molecule You would need to generate a primer that would sit on that single-stranded DNA molecule in a 5’ to 3’ fashion Bacterial enzymes will elongate and make DNA using the single-stranded DNA as a template When you have a few copies of the DNA, you can amplify using PCR to make many copies of that cDNA You can put this into a library by introducing DNA into bacterial vectors o Use an RT step, a poly-DT or poly-oligonucleotide to hybridize with the poly-A tail of all the molecules in the complex sample solution o Extend it with RT and remove the RNA by an alkali treatment or with RNAase o Leaves you with single-stranded DNA complementary copy to the RNA that you started with mRNA is converted to complementary DNA or cDNA by priming the poly A tail with a single-stranded poly T oligonucleotide RT uses this primer to initiate single-strand DNA synthesis that is fully complementary to the mRNA template RNA is then removed and a poly dG adapter (a small bit of DNA with a known sequence) is annealed to the 3’ end. A poly dC primer is used to initiate synthesis of the second DNA strand o Single-strand DNA molecule with unknown ends add on poly dG nucleotide to all of them o Use DNA ligase so you have a bunch of molecules with a poly dG end (3’ end) o Then you know the end so you can prime the second DNA synthesis step using a poly dC oligonucleotide that will recognize the bit added on o It will base pair with the adapter to prime second strand synthesis with E. coli DNA polymerase I so that you form a dsDNA molecule representative of the mRNA that was present in the initial source of sample E. coli DNA polymerase I progresses through any remaining hybrid regions and extends the second strand o The number of cDNA molecules that correspond to any given DNA product will also represent DNA abundance Studying genes of interest: Genomic and cDNA libraries cDNA libraries correspond to the population of mRNA molecules present at a given time, or in a given tissue (or both) Permanent, reusable record of the mRNA expressed in a given tissue/stage Every single mRNA (cDNA) should be represented at least once among the phage plaques complexity Probes allow you to find specific cDNAs – allows you to identify specific sequences of DNA based on hybridization, complementarity Polymerase Chain Reaction (PCR) A technique that has revolutionized molecular biology PCR has become feasible for routine lab work by the discovery of thermostable DNA polymerases o These were isolated form extreme thermophiles that live in very harsh conditions, i.e. the hot springs and geysers in Yellowstone National Park, Wyoming, USA PCR greatly facilitates the exponential amplification of target DNA molecules from a minimal amount of starting template, in the extreme case from a single molecule (the ice-man, forensic science, Neanderthal ancestors) Studying genes of interest: RT-PCR Since all mRNAs present in a given cell will have a poly-A tail Poly-T primers RT-PCR Abundance of cDNA is representative of transcript number cDNA: What to do with it Recombinant proteins can be overexpressed in E. coli to make large quantities of protein: insulin The lac promoter provides an effective means of inducing gene expression o Lac operon in bacteria turns on lac Z gene o Can take out lac Z gene and put any cDNA in its place (e.g. insulin, growth hormone) . Induction of lac gene expression can make tons of that cDNA Bacterial expression vectors: over-expression of recombinant protein Recombinant proteins can be overexpressed in E. coli to make large quantities of protein – insulin, hGH, EPO, interferon The lac promoter provides an effective means of inducing gene expression Lecture 18 – Molecular Genetics Methods II (continued) Specialized vectors permit efficient expression in higher eukaryotic cells Specific vectors often have a function – require elements important for transcription (promoters) Can use viral signals encoded into these vectors that will specify where to put the poly-A tail Don’t always want to express a protein in bacteria Can introduce expression vectors that express cDNAs into eukaryotic cells in a process similar to transformation in bacteria - Transfection Transient transfection: only some cells express the transgene o Vector express cDNA of interest for a short period of time until cells realize it and kick it out Stable transfection: all the cells