26. DNA technology Flashcards
What are the basic DNA technology techniques?
Explain PCR
- PCR - polymerase chain reaction
- Makes many copies of particular DNA sequence
- Template DNA (taken from a small sample) - DENATURATION: the template DNA heated for the double strands to separate and become single stranded
- Oligonucleotide primers (15-20 nucleotides) are added - complementary to the ends of the sequence which is amplified (bind o opposite ends becuse synthesis only in 5’ to 3’ direction)
- ANNEALING: the primers anneal to the opposite ends of the two strands
- POLYMERISATION: thermostable DNA polymerase (Taq polymerase - from extromophiles) is used to extend the primers using dNTPs - (Mg is required because it is a cofactor)
- Put the sample into thermocycler which regulates the temperature for the three steps in PCR: denaturation - primer annealing - polymerysation (elongation)
- The cycles repeated many times to copy DNA many times - multiplies exponentially
What are olinucleotide primers?
Short sections of DNA which bind to the ends of the DNA sequence which is amplified in PCR
Forward and reverse primers - because annealing happens 5’ to 3’ end
What is reverse transcriptase-PCR used for?
For converting RNA into DNA
PCR summary (components)
What is gel electrophoresis used for?
What is the mechanism of DNA gel electrophoresis?
- DNA has negative charge (phosphate groups) - moves in electric current to separate through the porous agarose gel - the smaller the DNA - the further it travells
- Put DNA at negative end of the electrophoresis chamber - moves towards the positive end
- Ethidium bromide is added to visualise the DNA fragments - incorporates into DNA - fluorescent under UV
- DNA ladder - gives a reference of DNA size
What are the components of gel electrophoresis ste up?
What is DNA sequencing used for?
To figure out the nucleotide sequence
What are the types of DNA sequencing?
Explain the mechanism of Sanger sequencing
PCR with fluorescent markers:
- In PCR dNTPs with coloured fluorescent marks added - stops when it is added - many short sequences - added up together can be wroked out the sequence of nucleotides
Explain the mechanism of next generation sequencing
- Also PCR used - sequencing by synthesis - adds one base at a time → removed
Explain the mechanism of third generation sequencing
- nano-technology used - single stranded DNA
- Example 1: DNA run through a nanopore - measure conductance across it - determine the abse sequence on conductance
What is DNA cloning used for
- Used to make many copies of DNA
- Difference with PCR: more stable, inside an organism - more accurate, as many copies as you want - in PCR limited copies, can get out RNA / protein, can make many copies of different DNA at the same time
What is the mechanism of DNA cloning
- sample of DNA taken - cut using restriction enzymes - ‘digestion’ at restriction sites (part of bacterial defence against viruses by cutting foreign DNA - own DNA protected by methylation)
- Restriction enzymes cut at palidromes between specific bases (regions where the sequence is read the same in both directions) → produces sticky ends (sometimes blunt but they ar enot as useful) → recombinant DNA from sticky ends
Explain palidromes
Explain plasmids as vectors structure
- At restriction site - restriction enzymes cut there and insert the recombinant DNA
- Secondary selective marker produces visual outcome from inserting new DNA (ex protein which produces blue colour will no longer be produced because the new DNA insert breaks the sequence → visual outcome that the DNA insertion occurred)
The mechanism of DNA insertion into a plasmid vector
- DNA and vector - cut at same sites with same appropriate restriction enzyme → sticky ends produced → gene of interest can be inserted into the plasmid
- DNA ligase seals the knicks → complete plasmid with the inserted gene
- Non-recombinant plasmid can still be produced if the sticky ends stick back together in the plasmid wothout the inserted gene (secondary selective marker would not show any change)
What is transformation and what is the mechanism of it
- Transformation - the mechanism of putting the vector (with recombinant DNA) into a host
- exposure to temperature shock makes the host take in the plasmid vector
What is selection and what is the mechanism of it
- When transformation is completed - the host bacteria are transferred onto plates with antibiotics → any bacteria that did not take the vector die
- Selected only the colonies with transformed bacteria
- Some colonies will show secondary selective marker (not innactivated) - those hosts took up the non-recombinant plasmids (without the desired gene - DNA insertion did not occur in those plasmids)
- Separate the recombinant plasmid hosts
Summary of basic DNA technology
