Genome Projects And Gene Technologies Flashcards
What is a proteome
All the proteins made by organism
Why is it easy to determine proteome of simple organisms ?
Don’t have much non coding DNA this is useful in medical research and development. E.g. identifying protein antigens on surface of disease causing bacteria and viruses can help with development of vaccines
Can you determine proteomes of complex organisms?
Yes but they have large sections of non coding DNA and contain complex regulatory genes ( which determine when genes are switched on or off) this makes it more difficult to translate their genome into their proteome as it’s hard to find bits of code for proteins among non coding and regulatory DNA.
What do you call organisms with transferred DNA
Transgenic organisms
How is reverse transcriptase used to make a DNA fragment ?
Reverse transcriptase - cells that produce protein coded for by target gene will contain many mRNA molecules that are complementary to the green ( mRNA is easier to obtain ) they are used as templates to make DNA, mRNA is isolated from cells then mixed with DNA nucleotides and reverse transcriptase which uses mRNA as a template to synthesis new strands of cDNA
How are restriction endonuclease enzymes used to make a DNA fragment ?
Some sections of DNA have a palindromic sequence of nucleotides so restriction nucleases are enzymes that recognize specific palindromic places and cut the DNA at these sequences because shape of recognition sequence is complementary to the enzymes active site. If recognition sequences are present at either side of the DNA fragment you want you the DNA sample is incubated out via hydrolysis reaction sometimes the cut leaves sticky ends which are used to anneal DNA fragment to another piece of DNA that has sticky ends with complementary sequence.
How is the gene machine used to make DNA fragments?
Sequence that is required is designed , first nucleotide in the sequence is fixed to a support eg a bead , nucleotides are added step by step in correct order in a cycle of processes that includes adding protecting groups ( these make sure nucleotides are joined at right points to prevent unwanted branching ), short sections of DNA called oligonucleotidesb( 20 nucs long ) are made they are broken off and protecting groups are removed so oligonucleotides can then be joined to make longer DNA fragments. Uses data base
What is in vivo and in vitro cloning ?
In vivo - gene copies are made within a living organism as organism grows and divides it replicates DNA creating multiple copies of gene
In vitro - where gene copies are made outside of a living organism using polymerase chain reaction
How is the DNA fragment using the target gene used in in vivo cloning ?( part 1 making recombinant DNA)
Vector DNA is isolated , vector DNA is cut open using restriction endonuclease that was used to isolate DNA fragment containing target gene so that sticky ends of vector DNA are complementary to sticky ends of DNA fragment containing the gene
Vector DNA and DNA fragment are mixed together with DNA ligase . DNA ligase joins sticky ends of DNA fragment to sticky ends of vector DNA ( this process is ligation ) , this new combination of bases in the DNA is recombinant DNA
What is the recombinant DNA used for in in vivo cloning
Vector with recombinant DNA is used to transfer gene into cells ( host cells ) that take up the vectors containing gene of interest are said to be transformed. If a plasmid is used host cells have to be persuaded to take in the plasmid vector and it’s DNA
How do you identify transformed cells ?
Only 5% of host cells will take up vector and it’s DNA . Marker genes can be inserted into vectors at same time as gene is cloned , host cells are grown of agar plates and each cell divides and replicates its DNA creating colony of cloned cells. Transformed cells will produce colonies where all the cells contain the cloned gene and the marker gene. Marker gene can code for AB resistance so host cells grown on agar played containing specific AB so only transformed cells that have marker will survive and grow or marker gene will grow under UV, identified transformed cells are allowed to grown more producing many copies.
How would you use in vivo cloning to produce proteins ?
Make sure vector contains specific promoter and terminator regions as without the right promoter region DNA fragment won’t be transcribed by host cells and protein won’t be made. Promoter and terminator regions may be present in vector DNA or may have to be added along with fragment
How are DNA fragments amplified using in vitro cloning ?
Reaction mixture set up that contains DNA , nucleotides , primers and DNA polymerase. Primers are short bits of DNA that are complementary to desired fragment.
DNA mixture heated to 95 to break H bonds mixture then cooled to 50 so primers can anneal
Mixture heated to 72 so DNA polymerase works nucleotides together then base pairing to complementary strand
Two new copies of fragment DNA are formed cycle starts again to get more
What are pros of transformed organisms
Crops can be given higher yields , higher resistance to pests or droughts so less chemicals needed , enzymes in industry can be produced , drugs and vaccines may be produced in large quants and cheap
What are concerns about transformed organisms ?
Crops genetically identical so all vulnerable to a disease and reduces biodiversity , superweeds , people junk they don’t have choice as they won’t know if their drugs were made genetically , worry that introduced toxins , smaller companies out of business , could be used to make designer babies ( illegal )
