Producing DNA fragment

What does recombinant DNA technology involve?

the transfer of fragments of DNA from one organism, or species, to another

What is recombinant DNA?

the DNA of two different organisms that have been combines

The resulting organism from recombinant DNA can be referred to as what?

transgenic

or

GMO - genetically modified organism

Since the genetic code is universal what does this mean for the transgenic organism?

the transcription and translation mechanisms are the same

the transferred DNA can be translated within cells of the recipient

What are the 3 ways fragments of DNA can be produced?

mRNA to cDNA using reverse transcriptase

using restriction enzymes to cut a fragmnet

creating the gene in a gene machine

What are the 5 key stages of producing transgenic organisms?

isolation of DNA fragments

insertion of DNA fragments into a vector

transformation - transfer of DNA into suitable host

Identification of host cells that have successfully taken yp the gene

growth/cloning

How is mRNA converted to cRNA?

mRNA isolated (cells are centrifuged)

free DNA nucleotides are added

reverse transcriptase joins DNA nucleotides to mRNA which acts as a template

enzyme (ribonuclease H) hydrolyses mRNA to get cRNA

is cRNA double or single-stranded?

single-stranded

What happens once we have cDNA?

DNA polymerase catalyses the formation of a complementary strand of DNA

produced a double strand (using free DNA nucleotides from earlier)

a copy of the gene now exists, with no introns

How are the cells chosen?

cell that readily produces the protein is selected

cells have large quantities of relevant mRNA which is more easily extracted

What do restriction endonuclease do?

cut up a DNA strand at a specific sequence of bases called a recognition sequence

Recognition sites are usually what?

palindromic (read the same left to right and right to left)

What are the different ways restriction endonuclease can cut up DNA?

some make clean cuts straight down - blunt ends

some make staggered cuts - sticky ends

What are oligonucleotides?

single strands of DNA

how does a gene machine work?

desired sequence of nucleotides can be determine by primary structure of protein

desired nucleotide base sequence is fed into a computer

the computer designs series of overlapping oligonucleotides which can be assembled into the desired gene

automated process assembles each of the oligonucleotides

oligonucleotides are joined together to make a gene - amplified by PCR

What happens once the gene is made?

using sticky ends, the gene can be inserted into a bacterial plasmid to be eventually cloned

Why can’t restriction enzymes be used to produce DNA that E.coli could use to produce a human gene?

human DNA contains introns

E.coli cannot remove introns

Why is it better to use the gene machine than reverse transcriptase to convert mRNA into cDNA?

faster to use gene machine than enzyme-controlled reactions

Why is the same restriction endonuclease used on each DNA sample when obtaining DNA fragments?

to cut at the same recognition site

to get required gene