Lecture 32 Recombination DNA Tech

sequences can be repaired with the highest priority

- composed of DNA binding Zinc finger protein and nuclease from the Fokl restriction enzyme
- Recognizes specific sequences on the right and left of the Fokl + and Fokl -

- composed of DNA binding transcription activatorlike effectors (TALEs) and the nuclease domain from Fokl
- Recognize single base pairs

- RNA guided nuclease that is derived from bacterial CRISPR-Cas9 system
- Recognizes a target DNA sequence relying on a short stretch of RNA
RNA sequence is a lot easier to make than ZFN and TALEN which require a lot of protein engineering to assemble

- RecA
- RecBCD
- Rad51

- catalyzes DNA synapsis
- facilitates the double strand break
1. RecA intertwined single strand and the duplex
2. single strand searches the duplex for homologous sequence
3: strand invasion: the single strand forms base-pairs with the complementary strand in the duplex (heteroduplex)

- recombination enzyme
- Helicase and nuclease degrade the DNA, suppressed when the CHI sequence is reached
- when CHI is reached, only the 5’ end is degraded, leaving a single stranded 3’ end

RecA, but in eukaryotes

a special DNA intermediate that contains 4 DNA strands shared between 2 DNA helices

- Heat strands to 94 degrees for 30 seconds to denature
- 50-60 degrees for 30 seconds for hybridization of primers
- 72 degrees for 1 minute for DNA synthesis from primers
● Multiple cycles of this amplifies the DNA fragment

- Refers to DNA sequencing
- Requires chain-terminating nucleotides as an essential reagent
- Dideoxynucleotides are required, which terminate DNA synthesis due to lack of the 3’ OH group
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