Gene Technology Flashcards
1st stage of PCR
The DNA to be amplified is heated to 95°c to break the hydrogen bonds between the nitrogenous bases
2nd stage of PCR
DNA is cooled to 40°C to allow the primers to anneal to each strand at specific points.
-insert what primers are and their functions-
3rd stage of PCR
The mixture is heated again to 70°C. DNA Taq polymerase copies each strand, starting at the primers and adding free nucleotides to form 2 new identical strands of DNA
What are primers (PCR)
Short chains, approximately 20 nucleotides long, which are complementary to the bases in the part of the DNA strand selected
What do primers do? (PCR)
1- they stop the 2 DNA strands from rejoining
2- they “bracket” the section of DNA to be copied
3- DNA replication can only be started in a double stranded region (which they provide)
What’s the role of PCR
Allows DNA to be amplified to produce sufficient quantities for forensic analysis
What is a DNA probe
A short strand of DNA (20 nucleotides long). It has a complementary sequence to the targeted section of DNA so that the bases will hybridise if that sequence is present
How probes are used to identify DNA fragments
1- section of DNA hydrolysed into sections using restriction endonucleases
2- sections are separated by gel electrophoresis
3- fragments transferred to nylon membrane
4- fluorescent probes added
5- wash off extra probes
6- hold under UV light
Obtaining donor DNA with restriction endonuclease
1- the R.E hydrolyses the bonds in the middle of the targeted section of the polynucleotide chain of donor DNA into smaller fragments
2- this can form blunt or sticky ends. Most from sticky which are more useful
3- ensure the vector is cut using the same R.E so that the sticky ends are complementary
Obtaining donor DNA with reverse transcriptase
1- the mRNA of the desired gene is isolated and extracted.
2- Reverse Transcriptase is used to make a single strand of complementary DNA, using the mRNA as a template and following normal base pairing rules
3- DNA polymerase is then used to make the double strand of the single stranded DNA
what is a restriction endonuclease
A highly specific enzyme that cuts double stranded DNA by hydrolysis at specific base sequences at either side of the desired gene
what is reverse transcription
the reverse of normal transcription. DNA is formed from mRNA rather than forming mRNA from DNA
How is donor DNA inserted into a vector
1- the plasmid is cut using the same R.E to create complementary sticky ends
2- the human DNA is annealed into the plasmid as complementary base pairing occurs and phosphodiester bonds between the backbones of the donor and plasmid DNA are formed by DNA ligase.
3- the plasmid DNA is now recombinant DNA and the plasmid can be referred to as a recombinant plasmid
Why are viruses good vectors
they are naturally adapted to shoot their genetic material into host cells
How are the vectors inserted into the host cell
1- if the host cells are incubated with calcium ions and subjected to heat shock, they will be more likely to take up the recombinant plasmids.
2- marker genes are used to identify which bacteria have successfully taken in the recombinant DNA, these are often sections of DNA that code for resistance to a specific antibiotic.
