Molecular Techniques in Medicine Flashcards
What are the pros/cons for using genomic DNA - lymphocytes for testing?
Pro: Same in all cells, used to investigate genetic mutations, promoter sequences, exon/intron junctions
Con:
Cannot provide information about tissue specific expression
What are the pros/cons for using mRNA (cDNA) from a tissue sample for testing?
Pro: can investigate tissue specific expression, different dev stages
Con: Not all genes expressed in all tissues. Cannot provide information about gene structure
What are the pros/cons for using proteins from a tissue sample for testing?
Used to investigate posttranslational processing - phosphorylation, proteolytic cleavage, etc.
How many bases are usually part of sequence specific cleavage?
4, 6, or 8 bases
All restriction sites are ________ over two strands.
Palindromes
What are “sticky ends”
Restriction cuts that leave 3’ or 5’ overhangs
What is the name of the original/classic restriction endonuclease?
EcoR1
What is the acronym for remembering northern/southern/western blots?
SNoW DRoP
What enzyme is used in PCR?
Taq Polymerase
What is the benefit of using a heat stable enzyme that doesn’t denature for PCR?
You can run it as a cycle, making many many copies
Why are microsatellites/Short tandem repeats (STR) helpful for forensics?
Microsatellites occur in exactly the same location in all humans, but the exact sequences are unique. 7 different sites = proof of identity
What does CRISPR stand for?
Clustered regularly interspaced palindromic repeats
How does CRISPR work?
When viral DNA from a bacteriophage is incorporated into bacterial DNA, it transcribes CRISPR RN which can be recognized by a Cas9 nuclease which will cause a double strand break. This works like human immunity in that the bacteria will forever be resistant to this virus.
What makes CRISPR cleavage so remarkable?
It makes a double stranded break in a very defined, very specific spot.
Is CRISPR capable of knock-out and knock-in?
Yes!
