Presentation on theme: "Un genio bizzarro Kary Banks Mullis nasce il 28 dicembre 1944 a Lenoir, North Carolina. Nel 1962 si diploma alla Dreher High School di Columbia, South."— Presentation transcript:
1Un genio bizzarroKary Banks Mullis nasce il 28 dicembre 1944 a Lenoir, North Carolina.Nel 1962 si diploma alla Dreher High School di Columbia, South Carolina.Nel 1966 consegue il master in chimica al Georgia institute of Thecnology.Nel 1972 consegue il Ph.D. in biochimica all’Università della California a Berkeley.Nel 1973 ottiene una borsa di studio post-dottorato in cardiologia pediatrica all’University of Kansas Medical School.Nel 1977 si trasferisce all’Università della California per occuparsi di chimica farmaceutica.Nel 1979 è assunto dalla Cetus Corporation di Emeryville, California, come chimico esperto del DNA.Nel 1983 inventa la Reazione a Catena della Polimerasi per la quale nel 1993 gli sarà conferito il premio Nobel per la chimica.Nel 1986 viene nominato direttore per la Biologia Molecolare della Xytronyx Inc. di San Diego (California).Attualmente è vicepresidente per la Biologia Molecolare presso la Burstein Techologies con sede ad Irvine in California.ReferenzeSaiki et al. (1985) Science 230:1350.Saiki et al. (1986) Nature 324:163.Mullis & Faloona (1987) Meth. Enzymol. 155:335.Erlish et al. (1988) Nature 331:461.Letture consigliateLE SCIENZE dossierN
2La grande intuizione di Kary Mullis DNA stampoprimer 1DNA polimerasi5’3’primer 15’3’primer 2DNA polimerasi5’3’5’3’5’DNA polimerasi3’5’5’3’
3The polymerase chain reaction (PCR) The standard PCR reaction, typically done in a volume of ml, contains:- DNA template ( copies)- Appropriate buffer (50 mM KCl, 10 mM Tris-HCl pH 8)- Magnesium (1,5-3 mM)- dNTPs ( mM each)- Primers bases in length (10-20 pmoles each)- Taq DNA polymerase (1-2 Units)DNA thermal cyclersT gradient
4Template DNA molecules with a constant-length end and a variable-length end
5Theoretical PCR amplification of a target fragment with increasing number of cycles N° = i x 2nwhere:i = initial number of DNA molecules (duplex DNA with constant 5’- and 3’-ends.)n = number of cycles
6Effect of Mg2+ concentration Primer selectionSelect primers with a random base distribution and when possible with reasonably high GC content.Avoid sequence with significant secondary structure.Check the primers against each other for complementarity.MgCl2 (mM)Effect of Mg2+ concentrationGenerally excess of Mg2+ will result in the accumulation of non-specific amplification products and insufficient Mg2+ will reduce the yield.1,8 Kb Units per 100 ml reactionEffect of Taq polymerase concentrationGenerally the optimum concentration of Taq polymerase is 1-4 units per 100 ml reaction. Increasing amounts can result in greater production of non-specific amplification products and reduced yield of the desired target fragment.110 bp
7Cycling ParametersDenaturation: DNA is denatured by briefly heating the samples to °C.Annealing: the primers are allowed to anneal to their complementary sequences by cooling to 40-60°C.Because of the very large molar excess of primers present in the reaction mix, hybridization occurs almost instantaneously and long incubation at the annealing temperature is not required (10-60 sec).The melting temperature (Tm) of a PCR primerC-G = 4°CA-T = 2°CA) 5’-GCGTTAGGCCAGCGGG-3’ Tm = 56°CB) 5’-CCATTTGAAATTTATA-3’ Tm = 38°CC) 5’-AGGTCCCCATCAGCGC-3’ Tm = 54°CExtension: the annealed primers are extended with Taq polymerase by heating to °C. The time of incubation varies according to the length of the target being amplified: allowing 1 min. for each kilobases of sequence is almost certainly excessive (Taq polymerase can add up to nucleotides per sec).
8Nested PCRIf the primers bind to more than one locus (e.g. paralog or common domain), then more than one segment of DNA will be amplified. To control for these possibilities, investigators often employ nested primers to ensure specificity.Nested PCR means that two pairs of PCR primers were used for a single locus. The first pair amplified the locus as seen in any PCR experiment. The second pair of primers (nested primers) bind within the first PCR product and produce a second PCR product that will be shorter than the first oneFirst set of primersandSecond set of primersandThe logic behind this strategy is that if the wrong locus were amplified by mistake, the probability is very low that it would also be amplified a second time by a second pair of primers.This procedure is designed to increase the sensitivity of PCR by directly re-amplifying the product from a primary PCR with a second PCR.
9Principali applicazioni della PCR Clonaggio di geni e screening di librerie genomicheManipolazioni e sequenziamento del DNADNA fingerprinting in medicina legale (test di paternità e di identificazione di reperti biologici)4) Ricerca di OGM e tracciabilità degli alimentiTest per rilevare infezioni batteriche e virali (HIV, Mycobacterium tubercolosis)Test per l’identificazione di oncogeni (ras)Studi di evoluzione molecolare (DNA antico)8) Studi del polimorfismo del DNA
10Agarose gel electrophoresis of DNA The gel slabs for DNA analysis are either agarose ( 0,6-2 %) or polyacrylamide (5-12 %)l DNA/HindIIIAnalysis of plasmid DNALSLanes 1,3 and 5 = undigested DNA (S)Lanes 2,4 and 6 = linearized DNA (L)
11When an electric current is passed through the gel, each fragment moves down it at a rate that is inversely related to the log of its molecular weight.Y = -0,026 X + 3,945Y = -1, ,945A DNA fragment migrated 49 mmantilogYY = 2,671469 bp
12Agarobiose basic repeating unit of AGAROSE Agarose is derived from various species of seaweed (Gracilaria or Gelidium). The agarobiose repeat unit forms long chains with an average molecular mass of Daltons. There are also charged groups present on the polysaccharide, most notably pyruvates and sulphates- The gelation process involves the formation of a macroporous matrix (the pore size nm varies with concentration and type of agarose).- Agarose gels are thermoreversible (Melting T = 93°C ; Gelling T °C)DNA can be readily detected at high sensitivity in a gel.The bands of DNA in the gel are stained with the intercalating dye ethidium bromide, and as little as ng of DNA in one band can be detected as visible fluorescence when the gel is illuminated with UV light ( 300 nm).EtBrEtBr