E1L7 - Recombinant DNA Techniques Flashcards
Give two methods to amplify a desired DNA strand
- PCR
2. Cloning
Ethidium Bromide
Intercalating agent that visualizes DNA on SDS-PAGE
Rank the following types of gels in increasing order of protein size: Agarose vs. Polyacrylamide vs. Pulsed-field gels
Polyacrylamide < Agarose < Pulsed-field
Do restriction enzymes cut RNA?
No, ds DNA.
Which techniques rely on complementary hybridization of a probe?
- Southern Blotting eg. EB Virus
- RLFP - sickle cell anemia
- FISH - e.g. Her2 breast cancer
- PCR - e.g. hemophilia
- Multiplex PCR - eg. Duchenne’s muscular dystrophy
- Microsatellite PCR - eg. forensics, maternity test
- RT-PCR - eg. H1N1
- Microarrays - eg. Oncotype Dx
MstII
Restriction enzyme site that’s mutated in sickle cell, resulting in a larger piece of DNA (uncut) 1.3 kB
Herceptin
Drug for breast cancer that is indicated by Her2 gene amplification (detected by FISH)
Steps of PCR cycle?
- Denaturation
- Annealing primers
- Extension
Can you do PCR on RNA?
No; you need two strands. You can use PCR if RNA is made into complementary cDNA first (double stranded).
Which is the palindrome of 5’-CGT * * * -3’ ?
A. CGTACG
B. GCATGC
A. CGTACG
(NOTE 1: they might not use 5’ to 3’ notation, but by convention, it’s 5’ to 3’ from L to R so make sure you flip it!
(NOTE 2: they don’t have to give you the other half of the palindrome for it to work - see the ***)
Explanation/trick:
5’-CGTACG-3’
3’-GCATGC-5’
So take the bottom strand and flip it over to get:
5’-CGTACG-3’
What disease results from mutating the Bcl 1 restriction site, and how do you detect it? Hint: don’t confuse Bcl1 with Mst2
Hemophilia, PCR
What disease results from mutating the Mst2 restriction site, and how do you detect it? Hint: don’t confuse with Bcl1
Sickle Cell Anemia, RFLP (restriction fragment length polymorphisms)
