The Nobel Prize in Chemistry 1980 for his fundamental studies of the biochemistry of nucleic acids, with particular regard to recombinant-DNA” Paul Berg 1/2 of the prize USA Stanford University Stanford, CA, USA b Berg was the first investigator to construct a recombinant-DNA molecule, i.e. a molecule containing parts of DNA from different species, ……. His pioneering experiment has resulted in the development of a new technology, often called genetic engineering or gene manipulation, which has already had important practical applications, e.g. the manufacture of human hormone with the aid of bacteria. Berg performed his experiment, however, as part of an incisive analysis of the chromosome of an ape virus (called SV 40) Viruses contain DNA (or sometimes RNA, another nucleic acid). …….

Usi delle DNA polimerasi nel laboratorio biomolecolare: -Esempi di marcatura del DNA: nick-translation -Amplificazione del DNA: PCR e sue applicazioni -Sequenziamento del DNA

Marcatura del DNA: Nick Translation (Dnasi)  - 32 P-dATP + dNTPs ****

Clonaggio del DNA: selezione ed amplificazione in vivo di geni o frammenti genici, inseriti in appropriati vettori di clonaggio PCR: selezione ed amplificazione in vitro di frammenti di DNA

La curva di amplificazione è iperbolica Rn =Aumento di fluorescenza rilevato durante la PCR (normalizzata in base al valore misurato al ciclo Ct) Ct =Il ciclo di “soglia”, in cui la fluorescenza comincia ad aumentare al di sopra del “background”.

PCR quantitativa automatizzata (Real Time) - II

Elettroforesi e sequenziamento del DNA

Tipi di primers per sequenziamento (nel vettore) “walking primer”

Sequenziatori di II generazione I tre sequenziatori automatici di DNA attualmente più diffusi: A.Sequenziatore “classico” a 96 capillari 9600 Applied Biosystems. B.Sequenziatore di II generazione 454 GS Roche. C.Sequenziatore di II generazione Illumina/Solexa A B C

Sample Input and Fragmentation The GS FLX and GS Junior Systems support the sequencing of samples from a wide variety of starting materials including genomic DNA, PCR products, BACs, and cDNA. Samples such as genomic DNA and BACs are fractionated into small, 300- to 800-basepair fragments. For smaller samples, such as small non-coding RNA or PCR amplicons, fragmentation is not required. Instead, short PCR products amplified using Genome Sequencer fusion primers can be used for immobilization onto DNA capture beads as shown below under "One Fragment = One Bead". The GS FLX and GS Junior Systems support multiple sample prep options, click here to see the full list. Library Preparation Using a series of standard molecular biology techniques, short adaptors (A and B) - specific for both the 3' and 5' ends - are added to each fragment. The adaptors are used for purification, amplification, and sequencing steps. Single-stranded fragments with A and B adaptors compose the sample library used for subsequent workflow steps. One Fragment = One Bead The single-stranded DNA library is immobilized onto specifically designed DNA Capture Beads. Each bead carries a unique single- stranded DNA library fragment. The bead-bound library is emulsified with amplification reagents in a water-in-oil mixture resulting in microreactors containing just one bead with one unique sample-library fragment.

emPCR (Emulsion PCR) Amplification Each unique sample library fragment is amplified within its own microreactor, excluding competing or contaminating sequences. Amplification of the entire fragment collection is done in parallel; for each fragment, this results in a copy number of several million per bead. Subsequently, the emulsion PCR is broken while the amplified fragments remain bound to their specific beads. One Bead = One Read The clonally amplified fragments are enriched manually or using the REM e System for liquid handling workstations. The fragments are then ready for loading onto a PicoTiterPlate device for sequencing. The diameter of the PicoTiterPlate wells allows for only one bead per well. After addition of sequencing enzymes, the fluidics subsystem of the sequencing instrument flows individual nucleotides in a fixed order across the hundreds of thousands of wells containing one bead each. Addition of one (or more) nucleotide(s) complementary to the template strand results in a chemiluminescent signal recorded by the CCD camera within the instrument. For a detailed explanation of this reaction see Sequencing Chemistry. Data Analysis The combination of signal intensity and positional information generated across the PicoTiterPlate device allows the software to determine the sequence of more than 1,000,000 individual reads with the GS FLX System and 100,000 individual reads with the GS Junior System, per 10-hour instrument run. For sequencing-data analysis, three different bioinformatics tools are available supporting the following applications: de novo assembly up to 400 megabases; resequencing genomes of any size; and amplicon variant detection by comparison with a known reference sequence.

APS: adenosine 5' phosphosulfate Pyrosequencing