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cDNA Cloning cDNA Klon

Learn about cDNA cloning. Saznajte više o cDNA klon.

cDNA Cloning cDNA Klon

A cDNA (short for complemantary DNA or copy DNA) is a DNA copy of an RNA, usually an mRNA. A cDNA (kratki za complemantary DNA ili kopija DNA) je DNA kopiju jedne RNA, obično jednom mRNA. Sometimes we want to make a cDNA library, a set of clones reprensenting as many as possible of the mRNAs in a given cell type at a given time. Ponekad želimo napraviti cDNA library, skup klonovi reprensenting što je više moguće od mRNAs u određenoj vrsti stanica u određenom vremenu. Such libraries can contain tens of thousands of different clones. Takve knjižnice može sadržavati nekoliko desetaka tisuća različitih klonovima. Other times, we want to make one particular cDNA- aclone containing a DNA copy of just one mRNA. Drugi puta, što želite napraviti jedan određeni cDNA-aclone sadrži DNA kopiju samo jedan mRNA. This technique we use depends in part on which of these goals we wish to achieve. Ova tehnika se koristiti ovisi u dijelu na koji od tih ciljeva koje želite postići.

cDNA Libraries cDNA Libraries

If we want to build a cDNA library, we can use a simple but effective strategy that relies on a process called nick translation. Ako želimo graditi cDNA library, možemo koristiti jednostavne, ali učinkovite strategije da se oslanja na procesu koji se zove nick translation. The essence of nick translation is the simultaneous removal of DNA ahead of a nick (a single-stranded DNA break) and synthesis of DNA behind the nick. Sustina nick simultani prijevod je uklanjanje DNA ispred jednog nick (jedan break-DNA) i sintezu DNA iza nick. The net result is to move, or translate the nick in the 5'-3' direction. Neto rezultat jest: premjestiti ili prevesti nick u 5'-3 'smjeru. The enzyme we usually use the nick translation is E.coli DNA polymerase I, which has a 5'-3' exonuclease activity that allows the enzyme to degrade DNA ahead of the nick as it moves along. U enzim se obično koriste nick prijevod je E.coli DNA polymerase I, koji ima 5'-3 'exonuclease aktivnost koja omogućuje da se razgraditi enzim DNA ispred i nick kao što se miče zajedno.

The central part any cDNA cloning procedure is synthesis of the cDNA from an mRNA template using reverse transcriptase. U središnjem dijelu cDNA klon bilo koji postupak je sinteza u cDNA iz mRNA predložak pomoću obrnutog transcriptase. Reverse transcriptase is like any other DNA-synthesizing enzyme is that it cannot initiate DNA synthesis without a primer. Reverse transcriptase je kao bilo koje druge DNA-synthesizing enzim je da se ne može pokrenuti sintezu DNA bez boja. To get around this problem, we take advantage of the poly(A) tail at the 3'-end of most eukaryotic mRNAs and use oligo(dT) as the primer. Da biste dobili oko ovog problema, smo iskoristili za poli (A) rep na 3'-kraju većina eukaryotic mRNAs koristiti i oligo (DT) kao fitilj. The oligo(dT) is complementary to poly(A), so it binds to the poly(A) at the 3'-end of the mRNA and primes DNA synthesis, using the mRNA as a template. The oligo (DT) je komplementarni na poli (A), tako da se veže na poli (A) na 3'-kraja mRNA i sinteze DNA primes, koristeći mRNA kao predložak.

After the mRNA has been copied, yielding a single-stranded DNA (the "first strand"), we partially degrade the mRNA with ribonuclease H (RNase H). Nakon mRNA je kopiran, popustljiv jednu DNA ( "prvi nasukavanje"), djelomično degradirati u mRNA s ribonuclease H (RNase H). This enzyme degrades the RNA strand of an RNA/DNA hybrid- just what we need to begin to digest the RNA from our first strand cDNA. Ovaj enzim degradirati i RNA obala jedna od RNA / DNA hibrid-samo što nam je potrebno kako bi se početi digest, RNA iz naše prvi nasukavanje cDNA. The remaining RNA fragments serve as primers for making the "second strand," using the first as the template. Preostalih ulomaka RNA služi kao početnice za izradu "drugi Strand," koristi kao prvi predložak. This is the nick-translation phase of the process and again, E. coli DNA polymerase I is the enzyme we use to carry out the nick-translation reaction. To je nick-prijevod faza procesa i opet, E. coli DNA polymerase sam je enzim ćemo koristiti kako bi provela nick-prijevod reakcija. The net result is a double-stranded cDNA with a small fragment of RNA at the 5'-end of the second strand. Neto rezultat je dvaput nasukan cDNA s malom fragment RNA na 5'-kraju drugog obala.
Our next task is to ligate the cDNA to a vector. Naš zadatak je da pored ligate u cDNA na vektor. This was easy with our pieces of genomic DNA cleaved with restriction enzymes, but cDNAs have no sticky ends. To je bilo lako s našim komada genocid DNA cleaved s ograničenjem enzimi, ali cDNAs nemate sticky završava. We can ligate blunt ends together, even though that is a relatively inefficient process. Možemo ligate tupim završava zajedno, čak i da je relativno neučinkovit proces. However, if we want the efficient ligation afforded by sticky ends, we can tack sticky ends (oligo[dC]) onto the cDNA, using an enzyme called terminal deoxynucleotidyl transferase (TdT) or simply terminal transferase and one of the deoxribonucleoside triphosphates. Međutim, ako želite efikasnu ligation afforded by sticky završava, mi može pričvrstiti sticky završava (oligo [dC]) na cDNA, koristeći enzim zvan terminal deoxynucleotidyl transferase (TdT) ili jednostavno terminal transferase i jedan od deoxribonucleoside triphosphates. In the case, we use dCTP. U slučaju, mi koristimo dCTP. The enzyme adds dCMPs, one at a time, to the 3'-ends of the cDNA. Dodaje se encim dCMPs, jednu po jednu, na 3'-kraja, cDNA. In the same way, we attach oligo(dG) ends to our vector and allow the oligo (dC)s to anneal to the oligo(dG)s. Na isti način, mi pridajemo oligo (dG) završava na naš vektor i dopustiti oligo (DC) s na žariti do oligo (dG) s. This brings the vector and cDNA together in a recombinant DNA that can be used directly for transformation. To dovodi u vektor i cDNA zajedno u rekombinantnim DNA koji se može koristiti izravno za transformaciju. The base pairing between the oligonucleotide tails is strong enough that no ligation is required before transformation. The base pairing između oligonucleotide tails je dovoljno jaka da ne ligation je dužan prije transformacije. The DNA ligase inside the transformed cells finally performs the ligation. DNA ligase unutar transformisane ćelije konačno provodi ligation.

See the DNA Molecule in 3-Dimensions Pogledajte Molekula DNA u 3-Dimenzije