1-basics of molecular biologybreve

8/9/2019 1-Basics of Molecular Biologybreve

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a = teosinte,

b = hybrid of teocinte and maize

c = today's hybrid maize

d = genetically modified sweet corn


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Biologia Molecolare:

La Biologia Molecolare è lo studio dei meccanismi che

regolano il funzionamento di una cellula: replicazione,

trascrizione e traduzione del materiale genetico.


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Componenti implicate

DNA

RNA

Protein


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• 2 filamenti formano una

doppia elica.

• I 2 filamenti corrono in

direzioni opposte (anti-

paralleli).

• Chimicamente, il DNA

consiste in 2 lunghi polimeridi nucleotidi, agganciati su

di uno scheletro di zuccheri

e gruppi fosfato.

DNA


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Gene : Unità ereditabile

• I segmenti di DNA che

trasportano l’informazione genetica sono chiamati geni.

• Corrispondono normalmentead un tratto di DNA che

codifica per una proteina oper una catena di RNA conuna funzione nell’organismo.

• Geni portano l’informazione

per la costruzione ed ilmantenimento delle cellule inun organismo e la trasmettonoalla progenie (trattogenetico).


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Trascrizione

• Trascrizione, produzione di un filamento di RNA

equivalente ad un tratto di DNA

• La trascrizione è il primo passo nell’espressione di un

gene.

• DNA RNA.

• Durante la trascrizione, il DNA viene letto dalla RNA

polimerase producendo un filamento di RNA conl’aggiunta di uracili (U) invece delle timine (T).

trascrizione

trascrizione inversa


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• mRNA porta l’informazione per la sequenza di una proteina

ai ribosomi, le fabbriche di sintesi delle proteine.

• Nell’mRNA ad ogni 3 nucleotidi (codone) corrisponde un

aminoacido.

• Nelle cellule eucariotiche, il precursore dell’mRNA (pre-

mRNA) dopo essere stato trascritto dal DNA, viene

processato a mRNA maturo grazie alla rimozione degli

introni—parti non-codificanti del pre-mRNA.

RNA Messaggero


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Proteine

• Proteine (polipeptidi) sono

formate da aminoacidi in una

catena lineare che si

organizza nello spazio in

forma globulare.

• La sequenza degli aminoacidi

in una proteina è definita dalla

sequenza del gene sulla basedel codice genetico.


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La traduzione è il primo passaggio nella biosintesi

delle proteine.


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Codice Genetico Degenerato


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Che cosa è il Genoma ?

• Il Genoma è l’intera raccolta dell’informazione genetica

di un organismo.

• Corrisponde normalmente a tutto il DNA o, per moltivirus, all’RNA.

• Il genome include sia i genes sia le regioni non-

codificanti del DNA.


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Tools

used inMolecular Biology


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Restriction Endonucleases

Enzymes recognize specific 4-8 bp sequences

Some enzymes cut in a staggered fashion - ―sticky ends‖

EcoRI 5’…GAATTC…3’

3’…CTTAAG…5’

Some enzymes cut in a direct fashion – ―blunt ends‖

PvuII 5’…CAGCTG…3’

3’…GTCGAC…5’


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Human DNA cleaved with Eco RICorn DNA cleaved with

Eco RI

5’-C-G-G-T-A-C-T-A-G-OH

3’-G-C-C-A-T-G-A-T-C-T-T-A-A -PO4

PO4- A-A-T-T-C-A-G-C-T-A-C-G-3’

HO-G-T-C-G-A-T-G-C-5’ +

5’-A-C-G-G-T-A-C-T-A-G A-A-T-T-C-A-G-C-T-A-C-G-3’

3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A G-T-C-G-A-T-G-C-5’

Complementary base pairing

+ DNA Ligase, + rATP

recombinant DNA molecule

5’-A-C-G-G-T-A-C-T-A-G- A-A-T-T-C-A-G-C-T-A-C-G-3’

3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A -G-T-C-G-A-T-G-C-5’

Restriction Enzymes for Transformation


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Cloning

• Digested fragments can be inserted intosmall circular DNA called plasmids

• The known sequence of the plasmid is knownand can be used to sequence the unknownDNA fragment


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Cloning

Bacteria will replicate plasmids in additionto their own genomic DNA


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Gel electrophoresis

• The basic principle is that DNA,

RNA, and proteins can all beseparated by means of an electric

field.

• In agarose gel electrophoresis,

DNA and RNA can be separated

on the basis of size by running the

DNA through an agarose gel.

• Proteins can be separated on the

basis of size by using an SDS-

PAGE gel, or on the basis of sizeand their electric charge by using

what is known as a 2D gel

electrophoresis.


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Macromolecule blotting & probing


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PCR

Template DNA

Heat to

break apart

Attach

Primers

Copy with

polymerase

Heat again to create

new templates


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PCR Analysis

The process follows the principle of DNA replication


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Trascrizione inversa

• Alcuni virus e i retrotrasposoni replicano il loro DNApartendo dall’RNA grazie alla trascrizione inversa

producendo copie di DNA (cDNA) dall’RNA


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Polymerase chain reaction (PCR)

• The polymerase chain reaction is an extremely

versatile technique for copying DNA.

• PCR allows a single DNA sequence to be

copied (millions of times), or altered inpredetermined ways.

• PCR has many variations, like reverse

transcription PCR (RT-PCR) for amplification of

RNA, and real-time PCR (QPCR) which allow for

quantitative measurement of DNA or RNA

molecules.


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PRIMER

• A primer is a strand of nucleic acid that serves as a

starting point for DNA synthesis.

• These primers are usually short, chemically synthesized

oligonucleotides, with a length of about twenty bases. They

are hybredized to a target DNA, which is then copied by

the polymerase.

• minimum primer length used in most applications is 18

nucleotides.

• Replication starts at the 3'-end of the primer, and copies

the opposite strand.

• In most cases of natural DNA replication, the primer for

DNA synthesis and replication is a short strand of RNA .


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Southern blotting

• Southern blot is a method for probing for the presence of aspecific DNA sequence within a DNA sample.

• DNA samples are separated by gel electrophoresis and then

transferred to a membrane by blotting via capillary action.

• The membrane is then exposed to a labeled DNA probe thathas a complement base sequence to the sequence on the

DNA of interest.

• less commonly used due to the capacity of other techniques,

such as PCR.

• Southern blotting are still used for some applications such as

measuring transgene copy number in transgenic mice, or in

the engineering of gene knockout embryonic stem cell lines.

Northern blotting


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Northern blotting

• The northern blot is used to study the expression patterns of a

specific type of RNA molecule as relative comparison among a set

of different samples of RNA.• RNA is separated based on size and is then transferred to a

membrane then probed with a labeled complement of a sequence of

interest.

• The results may be visualized through a variety of ways depending

on the label used. Most result in the revelation of bands representingthe sizes of the RNA detected in sample.

• The intensity of these bands is related to the amount of the target

RNA in the samples analyzed.

• It is used to study when and how much gene expression is occurring

by measuring how much of that RNA is present in different samples.

• one of the most basic tools for determining at what time, and under

what conditions, certain genes are expressed in living tissues.


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Western blotting

• In western blotting, proteins are first separated by size, in a thin gel

sandwiched between two glass plates in a technique known as SDS-PAGEsodium dodecyl sulphate polyacrylamide gel electrophoresis.

• The proteins in the gel are then transferred to a nitrocellulose, nylon or other

support membrane.

• This membrane probed with solutions of antibodies. Antibodies specifically

bind to the protein of interest & visualized by a variety of techniques,

including colored products, chemiluminescence, or autoradiography.

• Antibodies are labeled with enzymes. When a chemiluminescent substrateis exposed to the enzyme it allows detection.

• Using western blotting techniques allows not only detection but also

quantitative analysis.

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