Gene isolation and manipulation - regenerating recombinant DNA molecules
10 important questions on Gene isolation and manipulation - regenerating recombinant DNA molecules
What is needed to do a PCR?
- Free nucleotides, target DNA, primers, taq polymerase
- PCR reaction lasts several hours and consists out of 20-35 repeating cycles
- Cycle 1: heat up to 95 degrees C to denaturate the DNA by breaking the hydrogen bonds
- Reduce to 60 degrees C so primers can form hydrogen bonds
- Heat up to 72 degrees C so taq polymerase can start polymerisation
- After cycle 1 there are 2 copys of the target DNA (but they both still have flagging tails)
- Repeat
- After cycle 3 there are 8 copies of which 2 consist only out of target DNA
What is the target sequence after 25 cycles?
Reverse transcriptase is an enzym that can make dsDNA from mRNA. Why is the enzym called reverse transcriptase?
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Bacteria produce enzymes that recognize and cut DNA at specific palindromic sites (as a defence against virus DNA, such sequences in the bacterial genome are protected by methylation), a well-known example is the EcoRI enzyme of E.coli. What is the DNA sequence recognized by EcoRI?
Why is the recognition site (for bacteria to cut DNA) a DNA palindrome?
Name 3 general classes of cloning vectors:
- Plasmic vectors: small, circular, high copy number
- Bacteriophage vectors
- Vectors for larger DNA inserts
- Fosmids
- Bacterial artificial chromosome (BAC)
What is the key innovation in DNA polymerase property that made PCR technology possible?
Taq polymerase works well in a heated environment since it’s origination is in hot water pools. For PCR a high temperature is necessary to denaturate the DNA in the first place.
Where, in the procedure of cloning with a cloning vector should the ligation step be performed?
How can restriction enzyme recognition sites be created in a PCR-generated DNA-fragment?
Why would geneticists create restriction enzyme recognition sites in PCR-generated DNA fragments?
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