Chapter 21 - Recombinant DNA technology Flashcards
what does GMO stand for?
genetically modified organism
outline the rough sequence of stages involved with gene transfer
isolation insertion transformation identification growth/cloning
how may we produce DNA fragments (3)
- conversion of mRNA to cDNA using reverse transcriptase
- using restriction endonucleases to cut fragments from DNA
- creating the gene in a gene machine
outline the process of using reverse transcriptase to make cDNA (4)
- a cell that readily produces the protein is selected
- these cells contain large amounts of relevant mRNA
- reverse transcriptase is used to make DNA from RNA and this DNA is complimentary (cDNA) assembled using DNA nucleotides
- to make the second strand of DNA the enzyme DNA polymerase builds up complimentary nucleotides along the first cDNA template
where are restriction endonucleases extracted from?
specific bacteria which produce RE in order to cut up the DNA fragments of invading viruses
what is a sticky end
where the DNA is cut in a staggered fashion
outline the process by which genes are manufactured using the gene machine (7)
- determine amino acid sequence of the desired protein and DNA which is complimentary to mRNA is worked out
- the nucleotide base sequence are fed into the computer
- sequence checked to ensure it meets biosecurity standards and ethical guidelines
- the compute designs a series of small, overlapping single strands of nucleotides (oligonucleotides) to be assembled into the desired gene
- using an automated process each oligonucleotide nucleotide is added in the required sequence
- gene replicated using PCR (contains no introns) whihc also makes it double stranded due to complimentary nature
- inserted into plasmid for storage (these act as a vector)
define in vivo gene cloning
transfer of fragments to a host cell using a vector
define in vitro cloning
using the polymerase chain reaction
what is the importance of sticky ends?
if the same restriction endonuclease is used to cut DNA then all the fragments will have ends that are complimentary to one another meaning that any single stranded sticky end will be complimentary to any other
what steps must be taken to prepare a DNA fragment for insertion?
- must add a promoter region to the start of sequence in order for RNA polymerase to bind and begin transcription
- must add terminator region to the end in order to stop transcription
what is a vector?
a carrying unit for DNA used to transport DNA into a host cell
how are DNA fragments inserted into vectors?
the DNA fragments are mixed with the opened up plasmids and become incorporated due to complimentary sticky ends
how is the process of tranformation carried out?
modified plasmids are mixed together in a medium containing bacterial cells and calcium ions, Ca and temperature changes contribute making the bacterial membrane permeable and allowing the plasmids in
why might not every bacterial cell contain the modified DNA (plasmid)
- only a few bacterial cells take up the plasmid
- some plasmids will have closed up again without incorporating the DNA fragment
- sometimes the DNA fragments join together to form their own plasmid
how is it determined which bacterial cells have taken up the modified plasmids?
- some plasmids carry genes for resistance to more than one antibiotic
- bacterial cells which have taken up the plasmid will have resistance to a specific antibiotic and therefore will survive (this test which bacteria have taken up the plasmids)
- since the DNA fragment has been inserted into one of these sections which codes for resistance to a specific antibiotic, this antibiotic will kill bacteria with the modified plasmid
name 3 marker genes which can be used to determine whether a modified plasmid has been taken up by the bacteria
- antibiotic resistance
- make fluorescent proteins
- make enzyme which can be identified by process
what is the use of replica plating?
making multiple separate cultures of the same bacteria so that some can be tested for antibiotic resistance
