2 The Diploid Human Genome 46 chromosomes2 copies of a gene (or DNA sequence of interest)6 x 109 base pairs~ 6 pg (6 x g)Beta-globin gene is % of the entire genomeDystrophin (2.5 Mb) is 0.08% of the genome
3 General approaches for studying specific DNA sequences
4 DNA CloningGoal:Generate large amounts of pure DNA that can be manipulated and studied using a variety of different techniques.
5 RESTRICTION ENDONUCLEASES “the Molecular Scissors” The first major breakthrough for cell based DNA cloning was the discovery ofRESTRICTION ENDONUCLEASES“the Molecular Scissors”the 1978 Nobel Prize for Physiology or Medicine was awarded to Daniel Nathans, Werner Arber, and Hamilton Smith for their discovery of REs
6 RESTRICTION ENDONUCLEASES RECOGNIZE and CUT specific 4 – 6 bp PALINDROME sequences known as restriction sites5’- A G C T - 3’3’- T C G A - 5’5’- G A A T T C – 3’3’- C T T A A G – 5’AluIEcoRI
7 RESTRICTION ENDONUCLEASES Most restriction enzymes occur naturally in bacteria.Protect bacteria against viruses by cutting up viral DNA.Bacteria protects their own DNA from being cut up by methylation of restriction sites.More than 400 restriction enzymes have been isolated and are commercially available.
8 RESTRICTION ENDONUCLEASES A G C TT C G AAluIA GC TT CG ABlunt endsSticky endsG A A T T CC T T A A GEcoRIGA A T T CC T T A A
10 DNA Cloning: the steps Isolate DNA from organism Cut DNA with restriction enzymesLigate each piece of DNA into a cloning vector cut with the same restriction enzyme to create a recombinant DNA molecule.Transform recombinant DNA (cloning vector + DNA fragment) into a host that will replicate and transfer copies to progeny.
12 CLONING VECTORA small DNA molecule into which a another DNA fragment of an appropriate size can be integratedCan replicate independently of a host cell chromosomeProduces many identical copies of the inserted geneCarries at least one gene that gives it a selectable trait
13 Plasmid vectors have the following features Origin sequence (ori) required for replication.Selectable trait that enables E. coli that carry the plasmid to be separated from E. coli that do not (e.g., antibiotic resistance).Multiple cloning sites i.e., a large number of restriction sites in a small spaceSimple marker that allows you to distinguish plasmids that contain inserts from those that do not (e.g., lacZ+ gene)
15 Clone Selection using Blue/White screening Bacterial lacZ gene (b-galactosidase)b-galactosidase hydrolyzes a bond in a dye called X-gal, turning it blueThe cloning site for foreign DNA is in the lacZ geneDNA inserted = b-galactosidase inactive = White bacterial colonies in the presence of X-galDNA not inserted = b-galactosidase active = Blue bacterial colonies in the presence of X-gal
16 TransformationThe process whereby new DNA (such as a plasmid) is incorporated into a bacterial hostTreating bacteria with CaCl2Heat shock bacteria at 42oC followed by placing on iceTreating bacteria in a electric current (electroporation)
17 Possible outcomes of a cloning experiment Bacterium does not take up a plasmidBacterium takes up a non-recombinant plasmidBacterium takes up a recombinant plasmid
18 Grow bacteria on medium that contains ampicillin and X-gal
21 Genomic DNA libraries A collection of cloned DNA fragments obtained by the partial restriction digestion of the total DNA of an organismligation into an appropriate vectorReplication within the host bacteriaTwo typesgenomic DNA librariescDNA libraries
22 The average human genomic DNA library consists of ~75,000 independent clones
23 cDNA LibrariesFor the analysis of protein coding regions i.e., exons
24 Protein Expression Vectors Should be able to be transcribed and translated by the genetic machinery of the bacteria into which it is introducedPromoter for RNA polymeraseRibosomal binding siteTranscription terminator sequence
28 Polymerase Chain Reaction In vitro DNA cloning It is the SELECTIVE AMPLIFICATION of a single specific DNA sequence from within a heterogeneous mixture of DNA (usually whole genomic DNA)
29 Basic requirements of DNA replication PCR is DNA replication in a test tubeA DNA templatePrimersNucleotidesDNA polymeraseMgCl2Buffer
30 Prior information is required about the DNA sequence flanking the target sequence FLANKING SEQUENCETARGET SEQUENCEFLANKING SEQUENCE
31 PCR primersTwo primers are required for each PCR reaction, complimentary to opposite strands with their 3’ ends pointing towards each other
32 Properties of PCR primers Specific for the sequences flanking the target sequenceOptimally nucleotides longNo self complimentary regions within the primer OR regions of complimentary sequences between the two primers
33 PCR- the basic processSeries of cycles of three successive steps (30 sec – 1 min)DENATURATION OF DNAAt 95oCANNEALING OF PRIMERSFrom 50-70oCEXTENSION OF TEMPLATE(DNA synthesis)At 72oC30-35 cycles
34 PCR was revolutionized by isolating DNA polymerase from bacteria (Thermus aquateus) that live in hot water springs
36 DNA increases exponentially in each cycle The DNA of interest is amplified by a power of 2 for each PCR cycle6 cycles of PCR = 25 or 64 copies of DNA40 cycles of PCR = 240 or 1,099,511,627, or x 1012 copies of DNA!!!
37 PCR Template DNA + Forward primer & Reverse primer + dNTPs+MgCl2+Taq Polymerase2-3hrsAMPLIFIED PCR PRODUCT
39 Advantages of PCR Disadvantage of PCR Rapid and easy to performSensitive, amplification of DNA from minute samples is possibleRobust, making it possible to amplify DNA from degraded samples.Disadvantage of PCRPrior sequence knowledgeShort size range of amplification products100 bp bpChances of contamination