21.1 producing dna fragments Flashcards
what are the 5 processes of making proteins with recombinant dna technology?
1- isolation
2- insertion
3- transformation
4- identification
5- growth
in which 3 ways is the desired gene located/isolated?
1- mRNA -> cDNA by reverse transcriptase
2- restriction endonuclease cuts fragments containing desired gene in DNA
3- gene is created with a gene machine based on known protein structure
what type of viruses has reverse transcriptase?
retroviruses
what does cDNA stands for?
complementary DNA
how is the desired gene isolated by reverse transcriptase? (3)
1- the cell with the gene that could produce the desired protein is selected (which has large quantities of its mRNA)
2- reverse transcriptase is used to make cDNA (complementary DNA)
3- DNA polymerase makes 2nd DNA strand using cDNA
what are restriction endonucleases & what do they do?
defense enzymes that cut up viral DNA - recognise specific sequence of bases & cuts DNA at palidromes
what are palidromes?
6 base pairs as recognition site of restriction endonucleases
- could be read the same way
what are the 2 results of using restriction endonucleases?
1- blunt ends
- 2 opposite base pairs leaving 2 straight edges
2- sticky ends
- uneven cuts leaving exposed unpaired DNA bases
how is the gene machine used to seperate the isolated gene? (4)
1- base sequence is determined from the amino acid sequence
2- the base sequence is fed into computer & checked against international standards & ethical requirements for biosafety & biosecurity
3- oligonucleotides are designed & assembled into the desired gene
4- gene is replicated & amplified using PCR
what are oligonucleotides?
small, overlapping single strand of nucleotides
what are the advantages of using a gene machine?
1- made in short time
2- accurate
3- free from introns - transcribed & translated by prokaryotes
