Recombinant DNA IIcDNA clones are copies of mRNAsConstruction of cDNA clonescDNA: first strand synthesiscDNA: second strand synthesisLigate duplex cDNA into a plasmidLimitations of cDNA synthesisHow do you find a cDNA clone from the desired gene?Strategies for screening cDNA clonesScreening by hybridizationScreening for an expressed productExpression screening in eukaryotic cellsDifferential analysisDifferential analysis applied to muscle differentiationSubtractive hybridizationDifferential display of RT-PCR productsSequence everything, find function laterGenomic DNA clonesConstruction of libraries of genomic DNABAC vectors for large DNA insertsHow many clones make a representative library?Screening libraries of genomic clonesSequence everything: genomicsRecombinant DNA IIMaking, screening and analyzing cDNA clones Genomic DNA clonescDNA clones are copies of mRNAs•Much of the genomic DNA is not expressed as mRNA•Many issues about gene function are best addressed by examining the product that they encode.•The cDNA copies of mRNA contain primarily sequences that encode protein.•Therefore, cDNA clones are useful for many studies of gene function.Construction of cDNA clones•Use the enzyme reverse transcriptase to copy mRNA into complementary DNA, called cDNA. This is equivalent to the template strand of the duplex DNA.•Use a DNA polymerase to copy that cDNA into the nontemplate (message synonymous) strand.•Insert the duplex cDNA product into a cloning vector and propagate in a host, e.g. E. coli.cDNA: first strand synthesisProduct is complementary DNA, called cDNA. It is equivalent to the template strand of the duplex DNA.AAAAAAA5’3’mRNATTTTTAAAAAAA5’3’TTTTTAnneal oligo-dT primerReverse transcriptase:RNA-directed DNA polymeraseRNase HdNTPsTTTTTAAAAAAA5’3’Hydrolyze remaining RNA with baseTTTTT5’cDNA: second strand synthesisTTTTT 5’cDNAProblem: How to get a primer for 2nd strand synthesis?TTTTT 5’dCTPsTerminal deoxynucleotidyl transferaseCCCCTTTTT5’CCCCGGGGGGGGLigate an adaptor to the 3’ end5’3’5’3’TTTTT5’CCCCGGGG5’3’DNA polymeraseAAAAAdNTPsDuplex cDNA3’Ligate duplex cDNA into a plasmidTTTTT5’CCCCGGGG5’3’Duplex cDNA3’AAAAARestriction endonuclease Cut the adaptorTTTTTCCCCGGGG AAAAALigate duplex cDNA into a plasmidTransform the population of cDNA plasmids into bacteria.Result is a cDNA library.Limitations of cDNA synthesis•First strand synthesis often does not go to completion.–Individual cDNA clones will frequently have the reverse complement of only part of the mRNA.–Multiple cDNA clones from a single mRNA will be present in the library•Priming second strand synthesis is inefficient–Some methods necessarily result in the loss of sequences at the 5’ end of the nontemplate strandHow do you find a cDNA clone from the desired gene? •A cDNA library has >100,000 individual clones.•It contains copies of as many as 50,000 different mRNAs .•The frequency of occurrence of a cDNA from a given gene reflects the abundance of the mRNA for that gene.•Try to find correct 1 clone in about 100,000.Strategies for screening cDNA clones•Brute force screen for abundant cDNAs.•Hybridization with a gene-specific probe.•Express the cDNA in the host cell (i.e. make a functional protein product)–Specific antisera–Labeled ligand to a receptor–Assay for a function (complementation)•Differential analysisScreening by hybridizationEach bacterial colonies contains a single type of cDNA plasmidFilter replica of DNA in coloniesHybridize with a labeled DNA from gene of interestDetect by autoradiographyScreening for an expressed productFilter replica of protein in coloniesBind an antibody specific for the protein of interestDetect the bound antibody with an enzymatic assay (generating color or light).Expression screening in eukaryotic cellsExpression library:cDNA inserts in a vectorthat will drive expressionin eukaryotic cells+“transfect”Cell line that needs a cytokine (e.g. IL-3) to grow. Has no Epo receptor, will not grow in Epo.A transformed cell line that expresses the Epo receptor will now grow in Epo without IL-3. The plasmid with the Epo receptor cDNA can be isolated from this cell line.Epointroduce cDNAplasmids into cellsDifferential analysis•Instead of looking for one particular cDNA, look for cDNAs from all genes whose expression differs in the process under study–Differentiation from mesoderm to muscle–Response to different nutrients–Progression through S phase of the cell cycle•Methods: –Subtractive hybridization–Differential display–Hybridization to massively parallel arrays of cDNAs.Differential analysis applied to muscle differentiationmouse 10T1/2 cellsmultipotentialmyocytes (muscle)adipocytes (fat)chondrocytes (cartilage)5-azacytidineSubtractive hybridizationmRNA*cDNA (radiolabeled)mRNAanneal with mRNA in excessmRNA-*cDNA duplexes + *cDNA + mRNA HAP columnmRNA-*cDNA duplexes bind to column *cDNA + mRNA eluteUse the labeled *cDNA to hybridize to a library of cDNA clones from 10T1/2 -derived myocytesShould contain cDNAs for all mRNAs in common between the 2 cell typesShould contain cDNAs specific for cells differentiating into muscleIs unlabeled, will not interfere with subsequent steps.Repeat a few cycles10T1/2myocyteDifferential display of RT-PCR products•Make cDNA from all mRNA in the two different cellular states (RT = reverse transcriptase).•Use several sets of PCR primers to amplify a representative sample of all the cDNAs.•Resolve those RT- PCR products on a gel.•Find the products that are present in only one of the two cellular states being compared.•Try to isolate the corresponding gene.Sequence everything, find function later•Determine the sequence of hundreds of thousands of cDNA clones from libraries constructed from many different tissues and stages of development of organism of interest.•Initially, the sequences are partials, and are referred to as expressed sequence tags (ESTs).•Use these cDNAs in high-throughput screening and testing, e.g. expression microarrays (next presentation).Genomic DNA clones•Clones of genomic DNA contain fragments of chromosomal DNA. They are used to:–obtain detailed structures of genes –identify regulatory regions–map and analyze alterations to the genome, e.g. isolate genes that when mutated cause a hereditary disease –direct alterations in the genome–sequence the genome.Construction of libraries of genomic