amplifying DNA fragments Flashcards
Fragments of DNA can be amplified by
in vitro and in vivo techniques
what is the main in vitro method?
PCR (polymerase chain reaction)
what is the main in vivo method?
transferring the fragments to a host cell using a vector
why are the ‘sticky ends’ produced from restriction endonucleases important for in vivo amplification?
considerable importance because provided the same restriction enzyme is used (so same recognition sites are cut) we can combine the DNA of one organism with that of any other organism- once the complementary bases of the two ‘sticky ends’ have paired up an enzyme called DNA ligase is used to bind the phosphate-sugar framework of the two DNA sections to combine them as one
what are the main steps of in vivo amplification?
1- preparing DNA fragments for insertion
2- insertion of DNA fragment into a vector
3- introduction of DNA into host cells
4- marker genes
explain step 1 of in vivo amplification: preparding DNA fragment for insertion
- for transcription of any gene to take place RNA polymerase must attach to the DNA near a gene= promoter
- the nucleotide bases of the promoter attach both RNA polymerase and transcription factors= translation can begin
- so if we want our DNA fragment to transcribe mRNA in order to make a protein it is VITAL that it is attached to the necessary promoter region in order to start the process
- terminator region also needs to be added to other end of DNA fragment to stop transcription at the appropriate point
explain step 2 of in vivo amplification: insertion of DNA fragment into a vector
1- once an appropriate fragment of DNA has been cut from the rest of the DNA and the promoter and terminator regions added the next task is to add it to a carrying unit- a vecto, used to transport DNA into a host cell
2- most common type of vector used is the plasmid (circular lenghts of DNA found in bacteria that are separate from the main bacterial DNA). Plasmids most always contain genes for antibiotic resistance and restriction enzymes are used at one of these antibiotic resistance genes to break the plasmid loop
3- the restriction enzyme used is the same on that cut out the DNA fragment= ensures that sticky ends of the opened up plasmid are complementary to the sticky ends of the DNA fragment
4- when the DNA fragments are mixed with the opened-up plasmids, they may become incorporated into them- when they are incorporated the join is made permanent by using DNA ligase
5- these plasmids now have recombined DNA
regarding introduction of DNA into host cells, once the DNA has been incorporated into at least a few plasmids, they must then be reintroduced into bacteria cells- a process called
transformation
what does transformation involve?
- plasmids and bacteria cells being mixed together in a medium containing calcium ions- calcium ions and change in temperature make the bacterial membrane permeable allowing the plasmids to go through the cell-surface membrane into the cytoplasm
after transformation, why may not all the bacterial cells possess the DNA fragments with the desired gene for desired protein; some reasons ma be:
1- only a few bacterial cells, as few as 1% take up the plasmids when the two are mixed together
2- some plasmids will have closed up again without incorporating the DNA fragment
the task of finding out which bacterial cells have taken up the plasmids entails using the gene for antibiotic resistance which will be unaffected by the introduction of the new gene- process works as follows:
1- all bacterial cells are grown on a medium that contains antibiotic ampicillin
2- bacterial cells that have taken up the plasmids will have have acquired the gene for ampicillin resistance
3- these bacterial cells are able to break down the ampicillin and therefore survive
4- bacterial cells that have not taken up the plasmids will not be resistant to ampicillin and therefore die
after discovering which bacterial cells have taken up the plasmids, some cells will have taken up the plasmid and then closed up without incorporating the new gene which will also have survived, so the next task is to
identify the new cells without the new gene and eliminate them achieved using marker genes
there are a number of different ways of using marker genes to identify whether a gene has been taken up by the bacterial cells; they all involve the use of a second, seperate gene on the plasmid. This second gene is easily identifiable for one reason or another:
1- it may be resistant to an antibiotic
2- it may make a fluorescent protein that is easily seen
3- it may produce an enzyme whose action can be identified
explain use of antibiotic-resistance marker genes
- rather old method and been suspended by other methods
- to identify those cells with plasmids that have taken up the gene of interest a technique called replica plating is used
- the process uses the other antibiotic-resistance gene: the gene that was cut in order to incorporate the required gene e.g. resistance to tetracycline
- as this gene has been cut, it will no longer produce enzyme that breaks down tetracycline
- we can therefore identify these bacteria by growing them on a culture that contains tetracycline
explain how replica plating works
- take an absorbent cloth such as sterile velvet marked N,E,S,W and place onto the petri dish containing the bacteria (only some of the bacteria have taken up the gene of interest however) in the same orientation
- this will cause some of the bacteria to be transferred to the cloth which can then be placed onto tetracycline medium
- the ones that die therefore must have taken the gene of interest and so you can take those cultures from the original plate and use them to extract the protein produced from the gene of interest
explain use of fluorescent markers
- more recent and more rapid method is the transfer of a gene from a jellyfish into the plasmid that produces a green fluorescent protein (GFP)
- the gene to be cloned is transplanted into the cente of the GFP gene
- any bacterial cell that has taken up the plasmid with the gene of interest will not be able to produce GFP so bacterial cells that have NOT taken up the gene of interest will continue to produce GFP and fluoresce
what makes process of using fluorescent markers more rapid than use of antibiotic-resistance marker gene
- bacterial cells with desired gene not killed so no need for replica plating
- results can simply be obtained by simply viewing the cells under a microscope and retaining those that do not fluoresce = rapid process
explain use of enzyme markers
- another gene marker is the gene that produces the enzyme lactase
- lactase will turn a particular colourless substrate blue
- gene of interest is transplanted into the gene that makes lactase
- if a plasmid with the gene of interest is present in the bacterial cell the colonies grown from it will not produce lactase
- therefore when these bacterial cells are grown on the colourless substrate they will be unable to change its colour
define vector
simply a carrier- the term may refer to something like a plasmid which carries DNA into a cell
what does PCR do?
method of copying fragments of DNA (process=automated so is rapid and efficient)
what does PCR require?
- DNA fragment to be copied
- taq polymerase (obtained from bacteria in hot springs so is thermostable and does not denature at high temps)
- primers
- nucleotides
- thermocycler (a computer-controlled machine that varies temps precisely over a period of time)
what are primers?
short sequences of nucleotides that have a set of bases complementary to those at one end of each of the two DNA strands
what are the 3 main stages of PCR?
1- separation of DNA strand
2- addition (annealing of the primers)
3- synthesis of DNA
explain what happens in the first stage of PCR (separation of DNA strand)
- DNA fragments, primers and DNA polymerase are placed into a vessel in thermocycler
- temp is increased to 95 degrees celcius
- causing the two strands of DNA fragment to separate due to breaking of hydrogen bonds between 2 DNA strands
