Lesson 13: Recombinant DNA Flashcards
Do all living organisms use the central dogma?
For the most, all living organisms use the central dogma to make proteins (excluding some viruses).
What is recombinant DNA?
Combine DNA from different sources
When did recombinant DNA start being used?
Recombinant DNA was a breakthrough in biotechnology in the 1970’s.
What is an example of a use of recombinant DNA?
Initially, pigs were grown and their pancreas was harvested and insulin was isolated that way. However, once recombinant DNA was discovered, we were able to express insulin in bacteria and isolate it for medical use.
What are some major differences between prokaryotic and eukaryotic transcription?
Prokaryotes
- Promoter sequence is -35 and -10 nucleotides upstream from start site.
- Sigma factors bind to the promoter to recruit RNA polymerase (initiate transcription)
- Have NO introns
Eukaryotes
- Have introns
How do we account for the differences in prokaryotic and eukaryotic transcription in recombinant DNA?
- In the plasmid, include a bacterial promoter -35/-10 element
- Use a cDNA as the template strand because cDNA has no introns (exons only)
How is a cDNA made?
cDNA is made from a mature mRNA that has no introns (therefore the cDNA will have no introns)
1) Lyse cells and purify mRNA
2) Hybridize with poly T primer
3) Use mRNA sequence to make a DNA copy. REVERSE TRANSCRIPTASE adds to the 3’
Note: Reverse transcriptase goes from mRNA to DNA
4) You end up with a double stranded DNA at the end
How is DNA amplified in order to insert into plasmid?
PCR!
The steps are the same as we learned before.
1) Template sequence is cDNA
2) Heat separates the double stranded template DNA
3) Primers anneal to template strand (allowing DNA polymerase to extend from and synthesize new strand). Eliminates the need for primase, nuclease, and ligase
4) Enyzmes - DNA polymerase
5) Nucleotides
What elements do we need to include in the plasmid in order to carry out the functions of the bacteria?
1) Origin of replication - allows for replication of the plasmid
2) -35/-10 promoter - recruits RNA polymerase and promotes transcription of the inserted gene
3) Shine-Dalgarno sequence - recruits ribosome and promotes translation of the inserted gene
4) Multiple Cloning Site (MCS) - contains restriction sites for restriction enzymes that only cut the plasmid backbone once. This allows for insertion of new genes,
5) Selectable marker - retains the plasmid. Allows for the selection of bacteria that have taken up the recombinant plasmid.
- NOTE: since the plasmid is being placed in the bacteria we only need protein/RNA machinery for bacteria. However, the insert DNA is a human gene so a human protein amino acid sequence is made
Describe how a selectable marker works.
The bacteria does not take up all the plasmid, therefore a selectable marker is used to select for the bacteria that takes up the plasmid.
1) Pipette bacterial sample onto surface of agar plate.
2) Spread sample evenly over surface
3) Cells grow on surface
4) Drug is added and only cells that have the plasmid will grow (it has a drug resistance gene) and form a colony. The drug will kill the rest of the bacteria.
-> This is how the selectable marker selects the bacteria cells that have taken up the plasmid
What is transformation?
When bacteria changes based on its surroundings
What are the steps for molecular cloning/recombinant DNA?
1) PCR sequence of interest containing proper RESTRICTION SITES at the 5’ and 3’ end
2) Digest both the PCR sequence and the plasmid with the same restriction enzymes
3) Add the PCR sequence and plasmid together and let overhangs match up through complementary base pairing
4) Ligate the PCR product with the plasmid
5) TRANSFORM plasmid into bacteria cells.
- Incubate the plasmid in the bacterial cells. Then
warming them up and cooling it down allowing the
bacteria to take in the plasmid.
6) Plate bacterial cells onto petri plates with agar and drug to kill any cells that did not take up plasmid. Only the cells with the plasmid will grow, since the plasmid has a drug-resistance gene.
NOTE: This is all happening in a test tube
How do we check that our target gene is in the plasmid?
Gel electrophoresis!
- But first we need to cut the insert gene out of the plasmid.
How do we cut the insert gene out the plasmid?
Restriction enzymes!
- Can digest plasmid with restriction enzymes and note the sizes of the DNA fragments
How can we use gel electrophoresis to check that our target gene is in the plasmid?
We can run our plasmid in a gel that was cut with the restriction enzymes (so there will be different DNA fragment lengths)
- In order to run the plasmid across the gel:
- We would need electricity. This will cause the DNA to
run toward the positive electrode since the DNA
sugar-phosphate backbone is negatively charged.
Since this gel has pores, DNA fragments will stop at
different spots based on their lengths.- The smaller the fragment, the faster it will run.
Larger molecules can not travel as far.
- The smaller the fragment, the faster it will run.
- We would need electricity. This will cause the DNA to
- So then the gel electrophoresis will be visualized using a dye and using a ladder with known sizes we can compare the sizes we got to the ladder and it see if it matches the lengths that the DNA fragments are supposed to be.
