Creating DNA fragments Flashcards
(17 cards)
What are the three methods to creating DNA fragments
1) Reverse transcription
2) Restriction endonuclease
3) Gene Machine
How does reverse transcription work
Reverse transcriptase is used to make DNA copies from mRNA
1) A cell that naturally produces the protein of interest is selected
2) mRNA coding or the protein is in the cell , reverse transcriptase joins the DNA nucleotides with complimentary bases to the mRNA
3) Makes cDNA single stranded
4) DNA polymerase is used to make it double stranded
Advantage
Intron free because its copied from mRNA
Restriction endonuclease
RE are enzymes that cut up DNA ( natural in bacteria as a defence mechanism)
There are many RE an they all have complimentary shapes to different DNA base sequences ( recognition site )
Each enzyme cuts DNA at a specific location
Some enzymes cut at the same location in the double stranded Advantage Intron to create a blunt end
Other enzymes cut to create staggered ends ( HinIII) they’re palindromic and have sticky ends
Gene Machine
1) Examine protein to identify AA sequences and mRNA and DNA sequence
2) Enter DNA into computer which checks for biosafety and biosecurity
3) Computer creates small sections of overlapping single strands of nucleotides - oliglonucleotides
4) Oliglonucleotides are joined to create DNA for the gen5) PCR can amplify the quantity and makes it double stranded
How can the fragments be cloned to amply the sample
In vivo cloning
In vitro cloning
In vivo cloning
1) Insert DNA into vector
2) Transform host cell with vector
3) Identify transformed cells
4) Grow the host cell
Can only use DNA fragments made from a restriction endonuclease in In Vivo cloning
Fragments are modified by adding a promoter region or terminator region
How do we enter the fragment into a vector
Vector - carries the DNA fragment into the host cell ( usually plasmid )
1) Cut open the plasmid using the same RE used to create the fragment
2) Creates the same sticky end
3) When the plasmid and fragment is mixed complimentary base sequences align
4) Ligase anneals them
It catalyses the condensation reaction to form phosphodiester bonds between nucleotides
How do we transform the host cell to insert the vector
Make the cell more permeable by mixing with Ca2+ and heat shocking the cells
Vector is more likely to enter the hosts cytoplasm
What issues can occur during in vivo cloning
1) Plasmid didn’t get inside of the cell
2) Plasmid rejoins before DMA fragment enters
3) DNA fragment sticks to itself
How do we identify transformed cells
Marker genes - Can identify which bacteria took up the recombinant plasmid
1) Antibiotic resistance gene
2) Genes that code for fluorescent proteins
3) Gebrs that code for enzymes
Antibiotic resistance marker genes
Grow host cells on agar plates containing the specific antibiotic
Only transformed cells with the marker gene will survive and grow
Identified transformed calls are allowed to grow more producing copies of the cloned gene
Fl
In vitro cloning
Polymerase Chain Reaction
Amplify DNA fragment not in a living thing
How does PCR work
1) Create a mixture of DNA fragments , nucleotides , primers and DNA polymerase ( taq polymerase from bacteria )
2)Temp is increased to 95° to break hydrogen bonds
3)Decrease temp to 55° so primers can anneal
3) Increase temp to 75° so DNA polymerase can work making new strands
Advantages of PCR
1) Automated - efficient
2) Rapid - 100 billion copies of DNA made
3) Doesn’t require living cells - less complex techniques
What are DNA probes
Short single stranded DNA that is labelled radioactively or fluorescently
Used to locate alleles of genes and screening
How do we use DNA probes
Heat a sample of DNA from a patient to make it single stranded
Mix with DNA probes that are complimentary to alleles for specific genes
If the patient had the allele the DNA will bind with the probe and it can be identified using x-ray or UV light
