Lecture 18-19 Flashcards
Explain the required steps and components required for PCR
Steps- denature, anneal, elongate
Components- Template, primers, DNA polymerase, nNTP’s
Describe the DNA sequences recognized by and the function of restriction endonucleases.
Palindromic sequences, cuts at a specific place, sticky ends
Explain the principles of gel electrophoresis. What are the 2 gels
Uses electric current to separate based on charge, size, shape.
Agarose (Larger pore)
Polyacrylamide (smaller pore)
Explain Restriction Fragment Length Polymorphisms (RFLPs)
Cut DNA fragments with restriction enzyme, the lengths will be altered if disease is present
3 components of a plasmid vector- what is DNA ligase for
Origin of replication, antibiotic resistance gene, multiple cloning site.
Introducing DNA and cutting phosphodiester bonds.
What does the Sanger method use
dNTPs and ddNTP’s that lack -OH end
In order to perform PCR amplification of mRNA what enzyme is necessary
Reverse transcriptase
What is heterologous expression
Stuff made in bacteria or Humans (can have post-translational modifications)
What are CRISPR-Cas9 steps in order
SgRNA binds to percise sites on target DNA, PAM determines the sites od double strand breaks, Cas9 does the double strand breaks, donor DNA that houses mutation is inserted
Compare and contrast Southern blotting, northern blotting, western blotting, Southwestern,
ELISA.
Southern blotting: DNA
Northern blotting: RNA
Western blotting: Protein
Southwestern: Detects DNA binding probes and protein blotting
ELISA: Quantitate analyte and antibody levels
Describe FISH steps and how it can be used
Denature, hybridize, detect
Can detect deletions in chromosomes (DiGeorges syndrome)
Explain how the following microarrays can be applied in medicine:
3 things about producing recombinant DNA
