Ch 21 Flashcards
Name a limitation of using restriction nucleases?
Product still contains introns
Name a limitation of using a gene machine?
Need to know the amino acid sequence first
Name a limitation of using reverse transcriptase?
More time consuming and difficult steps
Describe what Recombinant DNA tech means
transfer of fragments of DNA from one organism to another
Name the 5 stages of producing clones of cells with a desired gene
1) Isolation of the DNA fragment
2) Insertion of DNA fragment into a vector
3) transformation of DNA into suitable host cells
4) identification of the host cells that have taken up the gene via gene markers
5) Growth/cloning of population of host cell
How can fragments of DNA be produced?
Conversion of RNA to DNA via reverse transcriptase
Use of restriction endonucleases of cut fragments containing desired genes
Use of gene machine
Describe how reverse transcriptase is used to make fragments of DNA
mRNA is isolated containing the desired gene and acts as a template from the reverse transcriptase to produce cDNA
DNA polymerase is used ti create double stranded DNA
Describe how restriction endonucleases are used to make fragments of DNA
Restriction endonucleases cut up viral DNA in bacteria at the recognition sequence to produce either blunt or sticky ends
Describe how the Gene machine works
AA sequence is determined as are mRNA codons and DNA triplets
Info is put on computer and checked for biosafety and biosecurity
Computer produces oligonucleotides and assembles these to produce a gene with no introns
What are oligonucleotides ?
small overlapping single strands of a nucleotide produced by a gene machine
What reaction is used to insert DNA into a vector
Polymer Chain reaction
What does ‘in vivo’ mean?
transfer fragments of a host cell via vector
What does ‘in vitro’ mean?
polymerase chain reaction
How do you prepare DNA fragment for insertion in in vivo gene cloning ?
Addition of extra lengths of DNA
Promoter = binding site of RNA polymerase
Terminator = site where RNA polymerase is released and trancitrpn ends
What are recognition sites?
sequences of DNA that are cut by restriction nucleases
Why use the same restriction endonucleases to cut DNA?
so fragment ends are complementary so ends are sticky and can later be joined by DNA ligase
What is the role of DNA ligase?
Bind sugar phosphate backbone together
How do you insert DNA fragment into a vector in in vivo gene cloning ?
Vector usually= plasmid
Endonucleases breaks the loop at an antibiotic resistant gene and DNA ligase incorporates them together
How do you introduce DNA into a host cell in in vivo gene cloning ?
plasmid and bacterial cell are put in medium with Ca2+
Ions and increased temp makes bacterial membrane more permeable so plasmid can move into eh bacterial cytoplasm
What is transformation?
introduce DNA into a host cell
Name three ways introducing DNA into a host cell can fail
only small amounts take up the plasmids
some plasmids don’t take up the DNA fragments
Some DNA fragments ends join to form its own plasmids
Name the three type of marker genes
Using antibiotic resistance
use of Fluorescent protein
Enzyme action that can be identified
Describe how replica plating works
Add gene to antibiotic resistant gene so those who die in antibiotic have taken up the gene. Also can uptake another resistance so can tell if bacteria taken up plasmid
Describe how GFP is used as a marker gene
no florescent = taken up gene
Describe how lactaase is used as a marker gene
Lactase turns colourless substrate blue.
If DNA fragment inserted in this gene then remain colourless shows it has been taken in
