Topic 6: Molecular Genetics Part 1 Flashcards
What do you have to do before studying RNA or DNA
Isolate it from the organism (extraction)
Method of DNA/RNA extraction depends on…
If you want genomic, plasmid, or organelle DNA
If you want total RNA or just mRNA
DIY method or use a kit
DIY method of DNA extraction
Phenol-chloroform
Steps of DNA extraction (phenol-chloroform)
- Homogenize aka grind/suspend cells
- Lysis (burst cells with detergent)
- Acidification (digest cell components)
- Extraction (phenol, chloroform)
Slide 5
Column based DNA extraction
Homogenize or suspend cells, burst cells (lysis), DNA binding to silica membrane, wash/let cell debris passed = pure DNA
Two methods of DNA extraction
Phenol-chloroform
Column based (model organisms)
Steps of mini-prep plasmid extraction
- Resuspend cells
- Lyse cells (high pH buffer)
- Neutralize w buffer, anneal together for 2-3 mins
- Separate cell debris
- Steps for column-based
Slide 7
In mini-prep, why return pH to neutral and allow DNA to anneal back together?
Destroys genomic DNA, leaves plasmid for collection
What is polymerase chain reaction
Put DNA polymerase in a test tube with everything in needs to synthesize DNA
What does DNA polymerase require to synthesize DNA in vitro (PCR)? (5)
- DNA template (from an extraction, etc, know a little ab sequence)
- Primers that complement the template (17-25 nucleotide DNA primers)
- dNTPs (DNA building blocks)
- MgCl2 (Mg cofactor of DNA polymerase)
- buffer solution (H2O+salt = env similar to inside cell)
Slide 9
PCR
Two key innovations in PCR
- DNA polymerase from Thermus aquaticus (Taq) was discovered: stable at high temps
- Development of automated thermocyclers
What does gel electrophoresis do
Separate DNA molecules on the basis of their size
How does gel electrophoresis work
DNA molecules exposed to an electric field migrate to positive pole due to negative charge phosphates on DNA backbone
Shape/size of DNA molecule determines the rate of migration (small ones go further)
What kind of gel is used in gel electrophoresis
Agarose or polyacrylamide
Two ways of visualizing DNA in a gel
- Ethidium bromide: intercalates between bases of nucleic acids, fluoresces red-orange in UV light
- SYBR-green: forms complex w DNA that absorbs blue light, emits green
Slides 13, 14
Visualizing DNA in gel
What are restriction enzymes
Enzymes that cut dsDNA at specific sequences called restriction sites
Naturally occurring in bacteria
What do bacteria use restriction enzymes for
RE (DNA endonucleases) used to destroy bacteriophage (virus that infect/destroy bacteria) chromosomes
What do REs generate
Sticky ends
Characteristics of the sequences REs cut at
4-8 bp sequences
Palindromic (read same forward as reverse)
Why don’t restriction enzymes cut their own host DNA
Methylation hides the RE site from the enzyme, only added to host cell chromosome (but foreign DNA with seq will be cut)
What is annealing
Single stranded nucleic acids naturally find complimentary nucleic acids to form double stranded nucleic acids
What is recombinant DNA
Nucleic acids from different sources brought together through annealing phenomena
2 main reasons we would want to use annealing in the lab
- Connect or join any two strands of DNA from diff sources (recombinant DNA)
- Find DNA among a sample that complements to a known piece of DNA (hybridization: allows us to identify unknown DNA)
Slides 18, 19
Recombinant DNA, hybridization
What is hybridization
Identification of unknown DNA through complementation
Sanger sequencing uses
DNA extraction, PCR, gel electrophoresis
How many PCR reactions in sanger sequencing?
Four
Each PCR in sanger sequencing contains the normal components plus…
small amount of one of four dideoxynucleotides (stops DNA synthesis)
Modern sanger sequencing uses…
Fluorescent labels on each ddNTP, reaction can be read in the same lane using a laser
