what does DNA recombinant technology involve?
the transfer of fragments of DNA from one organism or species, to another
Give na application of DNA recombinant technology
allow large quantities of pure proteins to be producde
give the 5 steps of using DNA recombinant technology
- isolation/ production
- insertion
- transformation
- identification
- growth/cloning
Describe the 5 steps of using DNA recombinant technology
- isolation/ production of DNA fragments that contaion the desired gene
- insertion opf the DNA fragment into a host cell
- transformation ie the transfer f DNA into suitable host cells
- identification of the host cells that have taken up the gene
- growth/cloning of the population of host cells
what makes obtaining fragments of DNA potentially difficult?
must find the gene then isolate it from millions of beases making up the gene
give some othe methods of obtaining DNA fragments
- enzymes caklled restriction endonucleases to cut fragments
- converting mRNA to cDNA using recerse transciptase
- creating the gene in a gene machine
define the term sticky ends
- single stranded sections of DNA
- they form an overhang at the end of a double stranded molecule
- these single stranded bases are exposed and can ben to a complementarty sticky end
decrivbe how do restriction endonucleases work
- cuts dna at a specific sequence of bases
- by breaking the phosphodiester bonds in the sugar phosphate backbone
what does palindromic mean?
the base sequenmce on one strand is the same sequence as on the other strand, but in reverse
Suggest two advantages of using reverse transcriptase to produce a gene in comparison to removing DNA from an organism’s genome using restriction endonucleases.
- mRNA is at a high concentration within cells therefore it easier to isolate compared to single DNA copy of a gene
- The cDNA produced will have the non-coding introns removed. The gene will therefore be able to be expressed in prokaryotes
why is the new molecule called cDNA when using reverse transcriptase?
the DNA molecule produced is complementary to the original mRNA molecule
What is the function of the DNA polymerase?
- It synthesises a second DNA strand using the single stranded cDNA as a template, to form double-stranded DNA.
- It forms the bonds that create the sugar phosphate backbone, joining the new nucleotides together.
If attempting to obtain the gene that codes for the hormone insulin, why
must pancreatic cells be used for this technique?
Only transcribe mRNA in these cells
What could be an advantage of producing artificial genes in this manner ie gene machine?
-The genes would be free of introns and other ‘non-coding’ DNA (so they can
transcribed and translated by prokaryotic cells)
-any sequence of nucleotiodes can be produced in a short period of time with great accuracy
How do bacteria reproduce?
Binary fission
What does ‘in vivo’ mean?
in living organisms / cells
What is a plasmid?
Small, circular DNA found in the cytoplasm of bacteria that is separate from the main DNA.
Why are plasmids particularly useful in gene technology?
Can be transferred into bacteria easily. May contain genes for antibiotic resistance
that can be used as a genetic marker. Contains a replication origin so can replicate
The same restriction enzyme is used to cut both the DNA fragment from the source
DNA and the DNA of the vector.
Why is this important?
Vector and fragment need to contain
complementary ‘sticky ends’ to allow them
to form a recombinant plasmid.
