What are the three ways to isolate a target gene?
1 - Restriction enzymes2 - Reverse transcriptase3 - Artificially synthesise a gene
How can restriction enzymes be used to isolate target genes?
- DNA contains palindromic sites- Restriction enzymes cut palindromic at specific recognition sites- If there is a recognition site either side of a target gene, restriction enzymes can be used to cut it out - Using a restriction enzyme will leave DNA with sticky ends (unpaired bases)
How can reverse transcriptase be used to isolate target genes?
- Cells only have two copies of each gene in their nucleus (hard to access)- Use an enzyme that reverses transcription- Form cDNA (complimentary DNA) outside of the nucleus
How can you artificially synthesise a gene?
- Use a gene machine to make DNA from scratch- Join around 25 nucleotides together at once- Forms an oligonucleotide- Oligonucleotides can be joined together to form a synthetic gene- Allows you to design your own genes/proteins
What are the three steps in inserting a target gene?
1 - Isolate the target gene2 - Insert the gene into a vector3 - Insert the vector into a bacteria
What is a vector?
Something that is used to move DNA from one place to another
What are 2 examples of vectors?
- Virus- Plasmid
How do you insert a gene into a vector?
- Cut the vector with the same restriction enzyme used to isolate the target gene so that the sticky ends are complimentary- DNA ligase will then form the phosphodiester bonds between the isolated gene and the vector- Recombinant DNA is formed
What is recombinant DNA?
DNA from more than one source/organism
How do you insert a vector into a bacteria?
- Increase the permeability of the bacterial cell wall by treating with calcium chloride and then heat shock it- Marker genes can then identify which bacteria became transgenic
What is a transgenic bacteria?
Bacteria containing recombinant DNA
What are marker genes?
Genes that are paired with target genes to check if the vector has been inserted properly
Why are marker genes used?
Vectors are often not taken up by bacteria so marker genes are used to identify which bacteria are transgenic so that we can select and culture them
Why are marker genes easily identified?
Can be antibiotic resistance, UV or radioactivity which are easily identifiable and distinguishable
What will transgenic bacteria contain?
- Marker gene- Target gene
What is the PCR?
In vitro DNA amplification
What is the PCR used for?
Used to amplify DNA
What are the ‘ingredients’ needed for the PCR?
- DNA sample- Free DNA nucleotides- Primers- DNA Polymerase
What are primers?
- Short sequences of DNA complimentary to the start of the DNA sample, they anneal to a short sequence of single stranded DNA to make it double stranded so DNA polymerase can attach- They select the section of DNA to be copied
What is the PCR method?
1 - Heat to 952 - Cool to 503 - Heat to 704 - Repeat
In PCR why do we initially heat the sample to 95?
To break the hydrogen bonds between DNA nucleotides and therefore separate DNA strands
In PCR why do we secondly cool to 50?
- Allows primers to anneal- DNA polymerase can then attach- Complimentary base pairing of primers can then give a short double stranded section of DNA
In PCR why do we finally heat up to 70?
- DNA polymerase adds complimentary free nucleotides- Form phosphodiester bonds
What are the six steps to obtaining many copies of a target gene which has been translated to proteins?
1 - Isolate the target gene using restriction enzymes 2 - Insert the gene into an appropriate vector (same restriction enzyme because of complimentary sticky ends)3 - Insert vector into bacteria to form a transgenic organism (force membrane permeability by ice cold calcium chloride and then heat shock)4 - Identify which organisms are transgenic ( marker genes)5 - Culture the transgenic organisms ( transcribe and translate recombinant DNA to make protein of the target gene)6 - Extract/purify the protein
