Molecular Techniques Flashcards
Restriction Endonuclease
Cuts in the middle of the DNA sequence. Cuts Palindromes; Cuts in the exact spot, cuts at phosphodiester bond
Palindromes
sequences that read the same in the 5’ to 3’ direction.
How do organisms avoid using restriction endonucleases to cut their own DNA?
DNA modification. E.coli methylates self DNA.
Type II restriction endonuclease
produce 5’ overhang
Types of Cloning Vectors
BACs and YACs, plasmids, bacteriophages
PCR Process
1) obtain DNA template 2) make complementary oligonucleotide primers 3) add primers (1000 fold) 4) heat DNA to make ssDNA 5) run DNA polymerase (TAq) 6) repeat heating, cooling, polymerase cycle. Note after cycle 3, target DNA available in high quantities. after 20 cycles, target 10^6 greater quantity.
Expression Vectors
Have everything normal vectors have, but also have promoter sequences, transcription termination sequences, , and a ribosome binding site.
DNA hybridization
mixtures of DNA ss; ones that have sequence homology illuminate. (application, to see if disease markers are present via hybridization and illumination.
DNA libraries
collection of plasmid vectors that each have recombinant DNA of the entire genome.
Colony hybridization
grow each cell that has recombinant DNA in a library; transfer cells to paper; lyse cell; denature DNA; hybridize with radioactive probe; wash and expose to X-ray film
Chain termination
dideoxy or Sanger’s method; 3’-OH critical for linking nucleotide units; takes advantage of this fact by removing OH
Sanger Sequencing process
1)make ssDNA 2) add primer 3) Add nucleotides a dideoxynucleotides 4)creates fragments of DNA stopping at dideoxy nucleotides 5) run gel and read from the bottom of the gel upward
Next-gen Sequencing
Illumina DNA sequencing;
