producing DNA fragments

recombinant DNA

the combined DNA of two different organisms resulting in a genetically modified organism

stages of making a protein

isolation of the DNA fragments that have the desired gene

insertion of the DNA fragment into a vector

transformation - transfer of DNA into host cells

identification of host cells that have successfully taken up gene by gene markers

growth or cloning of population of host cells

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 the gene in a gene machine based on known protein structure

process of using reverse transcriptase

B cells from islets of Langerhans make mRNA that codes for insulin

mRNA acts as a template to form single stranded cDNA using reverse transcriptase

cDNA is isolated by hydrolysis of mRNA with an enzyme

double stranded DNA is formed on the cDNA template using DNA polymerase

restriction endonucleases

restriction endonucleases cut DNA double strand at a specific recognition sequence

it can leave blunt ends or sticky ends depending on the cut of two strands

process of using a gene machine

desired sequence of nucleotide bases, then mRNA codons and complementary DNA triplets are determined

desired nucleotide sequence is given to computer which checks for biosafety and biosecurity

computer designs series of small overlapping single strands of nucleotides, oligonucleotides, which are assembled by adding one nucleotide at a time

oligonucleotides are joined to make a gene with no introns which replicates with PCR

PCR constructs and replicates complementary strand to make double stranded gene

using sticky ends the gene can be inserted into bacterial plasmid as a vector

the genes are checked and those with errors are rejected