8 genetic technology Flashcards
define recombinant DNA
DNA made by joining pieces from 2 or more different sources
write an overview on gene transfer
1- the required gene is identified
it may be cut from a chromosome, made from mRNA by reverse transcription, synthesizd by nucleotides
2- multiple copies of the genes are made by PCR
3- gene is inserted into a vector (plasmid, liposome, viruses)
4- vector inserts the gene into the cells
5- cells with new genes are identified and cloned
how can recombinant DNA be made
It is because of the universal genetic code that rDna can be formed
what do genetic engineers need
1- enzymes
restriction endonucleases, ligase, reverse transcriptase
2- vectors
plasmids, virus, liposomes
3- markers, genes that code for easily identifiable substances that can be used as markers, GFP ( glows under UV light) or GUS beta glucuronidase enzyme(colorless to colorl)
what is the role of restriction endonucleas in bacteria
is to restrict viral infection by recognizing + breaking down the DNA of bacteriophages, it can do this buy cutting the phage DNA into smaller pieces or cutting teh sugar phoshate backbone of DNA . it can give blunt or sticky ends.
how is bacterial DNA protected from restrictio endonuclease
by chemical markers or by not having target sites for resriction endonucleas to bind
state all the enzymes used and their roles
Restriction Endonucleases-
cuts the DNA strand so that the desired gene can be isolated or inserted into a vector
Reverse transcriptase: reverses transcription to form a single strand complementary DNA cDna FROM mRna coding for the desired gene
DNA polymerase: used to convert the single strand cDNA into double strand dna molecule of the desired gene
DNA ligase- used to insert the gene into the vector
cataylsyes the formation of phosphodiester bonds in the DNA sugar phophate backbone
what two enzymes are used in the preparatio of a plasmid vector
restriction endonucleases, DNA ligase
how is a plasmid prepared
1- human gene is isolated and treated with R.E whic cuts the DNA and forms stickt ends
2- bacteria is treated with R.E wghich cuts plasmid, creatig sticky ends
3- open plasmids and cut gene are mixed together, complimentary base pairing occurs in which sticky ends form H bonds with complimentary sequences
4- DNA ligase seals gap in sugar phosphat backbone, producing a closed cirle of doble stranded DNA containing the new gene , this is now recombinat DNA
how are recombinant plasmids transferred into bacteria
through a process called transformation, recombinant plasmids are mixed with bacteria, bacteria are put into a solution with high CA2+ ion concentraion and are given a heat shock (40 derees), some bacteria take up the palsmid and are said to be trsnformed. this is done to make the membrane mre permeable
what do plasmids that produce good vectors have?
circles of DNA- increase stability
small,low molecula mass, redily taken up by bacteria
an origin of replication- can be copied fast
have polylinkeres- short length of dna that have 2 or more sites for restricttion nzymes
have marker genes for screening, allows the cell that has taken up the plasmid to be identified
how is insulin produced using genetic engineering
Isolation of human gene:
mRNA that codes for insulin is taken from the pancreas from B-cells in islet of langerhans. this mRNA is incubated w reverse transcriptase. single stranded cDNA formed, using DNA polymerase doube stranded cDNA forms. this cDNA is treated with restriction E.NN preparation of vector:
bacterial plasmid is treated with the SAME restrictin E.N, to create sticky ends
MIXING OF DOUBE STRANDED CDNA WITH STICKY ENDS AND PLASMID WITH STICKY ENDS using DNA LIGASE
to produce recombinant plasmid which is introduced into teh bacteria . transformed bacteria is identified, gene is cloned by growing the bacterium
name some advantages of using insulin produced b genetic tech
- identiclal to human inslulin
- more rapid response
- cheaper to produce in large volume
- ethical and religious issues
what is a promoter and its uses
is the region of DNA which dtermines which gene will be expressed, it is the site where RNA polymerase binds to in ordeer to begin transcription
the promoter also ensures that RNA polymerase can recognize which is the DNA template strand
- regulates gene expression
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advanatages of flueroscent markers
- easier to identify
- more econmoical = time saving
- no resistance genes can be passed onto other acteria
disadvantages of antibiotic resistan ggenes
- risk of spreading resistan geneto other bacteria
- less effective during trasnformation
- require develeopmont of new antibiotics if no longer effective as markers
what is a marker
a marker is a gene that is transfered with the dsesired gene so that scientits can identify whch cells have been succesfully altered or contain recombinant DNa
how can antibiotic resistance spread to other bacteria
through transduction (bateria to vrus) or conjugation (bacteria to bacteria)
how to use GFP gene to identify trasformed
the GFP gene along w the desired gene are linked with a specific promoter, once promoter activatese adn protein is epressed, transformed bacteria will glow green under UV light
name some uses of PCR
since it is used to produce very large quantities of fragments of DNA and RNA from very small quantities, it can be used for DNA profiling to identiyffy criminals, or to determine paternity
what is necessary for PCR to occur
- target DNA, primers, DNA polymerase namel TAG polymerase, free nucleotides, buffer solution
why is tag polymerase used in PCR
it comes from a thermohphilic bacterium hence it does not denature at high temperatures involved during the firt step of PCR
its optimum temp is high enough to prevent annealing of the DNA STRANDS that have not been copied yet
outline the steps ofPCR
1- DENATURATION
2- ANNEALING
3- ELONGATION
1- double stranded dna is heated at 95degrees which breaks the H bonds that bond the two strands together
2- temperatue is decreased to about 65 so that primers can anneal to the ends of the single strands of DNA, by complimentary base pairing, primers redue rebinding of sperated strands
3- temp is incr to 72 which is the optimum temp for TAG polmeraw to create cDNA to produce the new identiacal doube stranded DNA
what is gel elctrophoresis
its a technqiue used widely in the analysis of DNA, RNA, protein. during elecrophres, the molecules are seperated due to their net ( overall) charge
