Recombinant DNA (Dr Curran) Flashcards
What is recombinant DNA?
• Recombinant DNA: Two or more DNA molecules from different origins united into a new combination of information
What is a clone?
• Clone: Multiple copies of the same genetic
information.
What are restriction enzymes?
-enzymes that restrict/cut dna molecules at specific bases
– Exquisitely specific
– Identical overhangs
1. USED IN CLONING SEGMENTS OF DNA INTO PLASMIDS.
2. USED TO IDENTIFY FRAGMENTS OF DNA
What are the three essential components to recombinant DNA?
• Plasmids – Origin of replication – Selectable mark – Transformable • Restriction Enzymes – Exquisitely specific – Identical overhangs • Ligase Enzyme -sticks DNA together
Describe the experiment that showcases how restriction enzymes are used in recombining segments of DNA into plasmids.
-stanley cohen and herbert boyer
-isolate and purify the strains 101 and 102
-colonies growing on a petri dish
-break open the cell (eg by harsh sound waves/sonification)
-isolate plasmid DNA by density gradient centrifugation
-digest plasma DNA with EcoRI:
+cut pSC101 with EcoR1, you get a linear fragment
+cut pSC102 with EcoR1, you get three fragments
-cut pSC101 and pSC102 open and put a 102 fragment in 101 (done by mixing them together then adding DNA ligase)
- put into a bacterial cell/ a grown Ecoli strain without plasmid treated with CaCl2 to make cells permeable
-place cells on growth medium
-select for sth that was TetR and kanR resistant
-when you cut DNA segments you end up with sticky ends (asymmetrically cut-some single strand DNA ready to bind)
How do you make sure the recombinant plasmid has the correct DNA fragments?
- gel electrophoresis
- agarose gel separates DNA fragments according to size (small DNA goes down faster)
- cut recombinant strain plasmid from experiment with EcoR1 once since new strain has two cutting site
- as many bands in gel as cutting sites
How do we create a recombinant with human DNA?
-HUMAN DNA DIGESTED WITH EcoR1
• So many bands.
• How to isolate the band we require? = we do an experiment:
-take a plasmid or other type of vector and cut it so we can have inserts, linearize them
-take human DNA and cut it randomly with EcoR1
-take vector(plasmid) with EcoR1 site mix with randomly cut human DNA and add ligase
-now we have a population of plasmids with human DNA segments
-take a plate and grow the transformed bacteria (can be ecoli) (plasmids) in ampicillin
-if colony appears white they have human DNA if it appears green they do not
-repeat experiment until high percentage of human DNA bacteria
How do we make a cDNA (copy DNA) probe?
- FIRST CATCH THE mRNA FROM YOUR SEQUENCE OF INTEREST
- can be extracted from red blood cell precursors
- HIJACK A RETROVIRAL ENZYME
- to use the reverse transcriptase and make cDNA from the mRNA
- USE DNA POLYMERASE TO MAKE THE SECOND STRAND OF DNA
- so you can add in a radioactive nucleotide
- create radioactive DNA that can be followed
- MAKE LOTS OF THE FRAGMENT
- insert cDNA into vector and make lots of copies
How do we identify the recombinant plasmid with the DNA segment we want?
- make a cDNA probe of the DNA segment we want
- use a filter to make an imprint of every colony on the plate we grew (we use a nitrocellulose disk which is like filter paper)
- lyse the cells (burst them open) to release the DNA from the cell
- treat the imprint in such a way that the DNA becomes a single strand and it glues to the filter (UV light)
- take labeled cDNA and flood it onto filter
- cDNA binds onto specific complementary sequence
- wash disk expose to xray film
- remove film, will show the wanted sequence as a black dot
- grow and isolate the wanted plasmid
- cut it with EcoR1 and gel electropheresis and now we have the isolated wanted sequence and it can be cloned
How do we use restriction enzymes to identify changes in DNA sequences?
-same experiment used to identify wanted DNA sequences can be done on gel electropheresis
-called southern blot:
• Agarose gel electrophoresis of digested DNA to separate fragments.
• Denature and transfer to a filter.
• Take cloned gene fragment and radioactively label it.
• Add to filter to allow hybridisation to occur.
• Lay onto photographic film to visualise
bands.
-OR we can use diagnostic cuts:
-sickle cell mutation- MST2 cuts normal gene, but not the mutation
-mutation will give one band, normal will give two bands
-gel electropheresis will then show two bands if normal, and one bands if mutated
-probe with cDNA
How do we clone without cells?
-Polymerase chain reaction (PCR)
-amplify DNA without cells
– Based on previously determined DNA sequence, develop short oligonucleotides (~20bp) complementary to sequences flanking the target DNA.
– Oligonucleotides act as primers to copy DNA similar to DNA replication.
– Each cycle of replication doubles amount of target DNA.
-necessary to know the DNA sequence close to ur target to do this
-denature, add primer, then add taq polymerase
What are the uses for PCR?
• Genetic mapping
• Genotype detection
• Analyze traces of partially degraded DNA
• Evolutionary studies
– Compare homologous sequences from related
organisms
– Compare sequences from a variety of sources
– Studies of gene diversity
• Diagnosis of infectious diseases
