Topic 8—B: Genome Projects and Gene Technologies- 2. Making DNA fragments Flashcards
What does recombinant DNA technology allow us to combine?
- Genetic material from different sources
What is the first step in recombinant DNA technology?
- Making a DNA fragment
What is a DNA fragment?
- A bit of DNA containing a gene
What does recombinant DNA technology involve?
- It involves transferring a fragment of DNA from one organism to another
How can the transferred DNA be used to produce a protein in the cells of the recipient organism?
- Genetic code is universal
- Transcription and translation mechanisms are pretty similar too
Recipient and donor organisms don’t have to be from the same species
- This can be useful
Genetic code (universal)
- The same DNA base triplets code for the same amino acids in all living things)
Transgenic organisms
- Organisms that contain transferred DNA
To transfer a gene from one organism to another what do you first need to get?
- A DNA fragment containing the gene you’re interested in (target gene)
What are the 3 ways that DNA fragments can be produced?
- Using reverse transcriptase
- Using restriction endonuclease enzymes
- Using a gene machine
Using reverse transcriptase
- Most cells only contain two copies of each gene, making it difficult to obtain a DNA fragment containing the target gene
- But cells that produce the protein coded for by the target gene will contain many mRNA molecules that are complementary to the gene- so mRNA is often easier to obtain
- mRNA molecules can be used as templates to make lots of DNA
- The enzyme, reverse transcriptase, makes DNA from an RNA template
- The DNA produced is called complementary DNA
Example (using reverse transcriptase)
- pancreatic cells produce the protein insulin
- they have loads of mRNA molecules complementary to the insulin gene
- so reverse transcriptase can be used to make cDNA from the insulin mRNA
How do you make cDNA? (Complementary DNA)
- mRNA is first isolated from cells
- then its mixed with free DNA nucleotides and reverse transcriptase
- the reverse transcriptase uses the mRNA as a template to synthesise new strands of cDNA
Using restriction endonuclease enzymes
- Most sections of DNA have palindromic sequences of nucleotides
- These sequences consist of anti parallel base pairs ( base pairs that read the same in opposite directions )
- restriction endonuclease are enzymes that recognise specific palindromic sequences (known as recognition sequences) and cut (digest) the DNA at these places
- Different restriction endonucleases cut at different specific recognition sequences, because the shape of the recognition sequence is complementary to the enzymes active site
- If recognition sequences are present at either side of the DNA fragment you want, you can use restriction endonucleases to separate it from the rest of the DNA
- The DNA sample is incubated with the specific restriction endonucleases which cuts the DNA fragment out via a hydrolysis reaction
- Sometimes the cut leaves sticky ends (small tails of unpaired bases at each end of the fragment)
- sticky ends can be used to bind (anneal) the DNA fragment to another piece of DNA that has sticky ends with complementary sequences
Using a gene machine
A database contains the necessary information to produce the DNA fragment
- This means that the DNA sequence does not have to exist naturally- any sequence can be made
Here’s how its done:
- the sequence that is required is designed (if one doesn’t already exist)
- the first nucleotide in the sequence is fixed to some sort of support e.g. a bead
- nucleotides are added step by step in the correct order, in a cycle of processes that includes adding protecting groups. Protecting groups make sure that the nucleotides are joined at the right points, to prevent unwanted branching
- short sections of DNA called oligonucleotides, roughy 20 nucleotides long, are produced. Once these are complete, they are broken off from the support and all the protecting groups are removed
- this oligonucleotides can then be joined together to make longer DNA fragments
