21 DNA technology Flashcards
what is recombinant DNA?
DNA of two different organisms that has been combined
process of making a protein using DNA technology
1) isolation: of DNA fragments that gave gene for desired protein
2) insertion: of DNA fragment into vector
3) transformation: transfer of DNA into suitable host cells
4) identification: of host cells that have taken up gene using gene markers
5) growth/cloning: of host cells
what are methods of producing DNA fragments?
conversion of mRNA to cDNA using reverse transcriptase
using restriction endonucleases to cut fragments containing desired gene from DNA
creating gene in a gene machine, based on known protein structure
how is reverse transcriptase used to isolate a gene?
cell that produces protein is selected which has lots of relevant mRNA so more easily extracted
reverse transcriptase joins DNA nucleotides with complementary bases on mRNA sequence (single stranded cDNA)
DNA polymerase makes other strand of DNA by building up complementary nucleotides on cDNA template forming required gene
how are restriction endoculeases used to produce DNA fragments?
cut DNA double strand at recognition sequence
what are two ways restriction endoculeses can cut DNA?
straight cut: between two opposite base pairs leaving blunt ends
staggered cut: leaving exposed DNA and sticky ends
how is the gene machine used to produce DNA fragments?
1) desired amino acid sequence determined, from this mRNA codons looked up and complementary DNA
2) desired sequence of nucleotide bases for gene is fed into computer and checked for biosafety
3) computer designs single strands of nucleotides (oligonucleotides) which are assembled into desired gene
4) replicated using PCR which also produces complementary strand to make double stranded gene
5) using sticky ends, gene can be inserted into bacterial plasmid which is vector
advantages of using the gene machine?
any sequence of nucleotides can be produced in a short time with great accuracy
no introns so can be transcribed and translated by prokaryotic cells
importance of sticky ends
if same restriction endonucleases used to cut DNA, fragments will be complementary and can be joined together
can be used to combine DNA of one organism with that of any other organism
what is DNA ligase used for?
once complementary stick ends have paired up, it binds phosphate-sugar framework of two sections of DNA
how is DNA fragment prepared for insertion?
addition of extra lengths of DNA
RNA polymerase binds to promoter region, nucleotide bases of promoter region attach RNA polymerase and transcription factors and begin process of transcription
terminator releases RNA polymerase and ends transcription
how is DNA fragment inserted into a vector?
same restriction endonuclease cut plasmid so sticky ends complementary to DNA fragment
joined together with DNA ligase forming recombinant DNA
how is DNA introduced into host cells?
transformation
plasmids and bacterial cells mixed in medium containing calcium ions and changes in temperature which make bacterial membrane permeable
why do not all bacterial cells possess DNA fragments with desired gene?
only few bacterial cells take up plasmids
some plasmids close up without incorporating DNA fragment
some DNA fragment ends join together to form own plasmid
what types of marker genes can be used?
antibiotic resistant marker genes - replica painting
fluorescent markers - GFP
enzyme markers - e.g. lactase, colour change
