gene technologies Flashcards
proteome
the full set of proteins produced by the genome.
recombinant DNA technology
the transfer of fragments of DNA from one organism, or species, to another.
name 3 ways to identify and isolate a gene from the rest of the DNA
- reverse transcriptase
- restriction endonuclease
- the gene machine
Reverse transcriptase
conversion of RNA into DNA.
- cell expressing a lot of the required protein is identified and isolated (e.g. B cells).
- the mRNA coding for the protein is extracted. It then acts as a template for the enzyme reverse transcriptase which creates a complemteary strand of DNA (cDNA).
- however the cDNA is single stranded and so the enzyme DNA polymerase is used to build up the other complimentary strand. = dsDNA of required gene (including introns).
restriction endonuclease
A class of enzyme that recognises and cuts DNA at a specific sequence of bases. The specific sequence of bases at which the restriction endonuclease cuts at is called a Recognition sequence. If this is between two opposite base pairs = 'blunt ends'. If it has a staggered line of cut = 'sticky ends' leaving both ends with some exposed bases.
the gene machine
- using bioinformatics the desired protein is used to determine its amino acids sequence and then its mRNA codons and so its DNA triplets.
- DNA triplets sequence is fed into a computer, which carrier out bio-saftey/security checks.
- a computer designs a series of small overlapping single strands called Oligonucleotides which can be put together to assemble the gene.
- the oligonucleotides are joined together (overlapping) one nucleotide at a time, creating the complete single stranded gene (containing no introns).
- using sticky ends the gene can be inserted into a bacterial plasmid and act as a vector for cloning, and the genes can be extracted from the plasmids and sequenced. Any errors are rejected.
in vitro cloning
Polymerase chain reaction (PCR)
DNA fragments, primers and DNA polymerase are placed in a thermocycler.
1. temperature is increased to 90 degrees C for 30 seconds. This breaks the h bonds holding the DNA strands together so they separate.
2. mixture is then cooled to 55 degrees C allowing the primers to anneal (bind) to the ends of the DNA strands by forming hydrogen bonds. This firstly prevents the ds reforming and secondly creates a starting point for DNA polymerase.
3. temperature is increased to 72 degrees for about a minute. This is the optimum temperature for DNA polymerase (taq). The DNA polymerase adds bases to the primer building up a complementary strand of DNA, producing a ds DNA identical to the original.
in vivo cloning
- isolation of gene using a restriction endonuclease (with STICKY ENDS).
- promotor region and terminator region must be added to the start and end of the DNA fragment.
- plasmid extracted from bacteria. Using the same restriction endonuclease cut the plasmid, producing complementary sticky ends to the target DNA.
- the enzyme DNA ligase is used to bind the sugar phosphate backbones by condensation reaction (phosphodiester bonds).
- the plasmids are reincorporated into bacterial cells, by mixing the plasmids in a medium also containing calcium or chloride ions (and an increase in temperature). This makes the cell membrane of the bacterial cells more permeable allowing the plasmids to pass in easier.
- identification: eg. using the gene for antibiotic resistance, by growing the bacteria on a medium containing the antibiotic. also using marker genes. You could also use florescent markers.
name of the technique used if in in vivo cloning the antibiotic resistance is used as an identification technique
replica plating. A sample of each gene colony is tested with the antibiotic. It is done by pressing a sterile cloth against the plate and pressing this onto the new plate. This ensures that the bacterial colonies are on the same area as they were on the first plate so they can be easily identified.
using florescent markers in in vivo replication
the Green florescent protein (GFP) is found in jelly fish.
advantages of in vivo cloning
- very accurate compared to PCR, as few errors in the DNA copied. (in PCR about 20% of the DNA is copied inaccurately).
- involves no risk of contamination, as due to the restriction endonuclease, anything not cut by the same enzyme will not have complimentary sticky ends and so cannot be incorporated.
advantages of PCR
rapid and does not require living cells.
DNA sequencing
This is the process of determining the sequence of nucleotide bases in a piece of DNA.
Sanger sequencing / the chain termination method
relies on dideoxy nucleotides. It will have one of the 4 different bases attached to it, and each will be labeled with a different coloured dye so you can distinguish between the different bases.
- DNA is heated to break the hydrogen bonds
- cool so a primer can bind to each single strand
- heat again so DNA polymerase can synthesis new DNA
- Nucleotides added to the template strand until a didexoy nucleotide is added, preventing any more from joining.
- repeated a number of times to guarantee that a dideoxy ribose nucleotide has been incorporated into every position on the target DNA at least once.
- gel electrophoresis. a laser and computer at the end detects the colour of each dye as it crosses. From this data the base sequence can be deduced.
what is a dideoxy nucleotide
similar to regular nucleotides but have no hydroxyl groups on the 3’ carbon on the sugar. This means that no further nucleotides can be added (why its also known as a chain terminator).
