Topic 6: Molecular Genetics Part 1

Question 1

What do you have to do before studying RNA or DNA

Answer 1

Isolate it from the organism (extraction)

Question 2

Method of DNA/RNA extraction depends on…

Answer 2

If you want genomic, plasmid, or organelle DNA
If you want total RNA or just mRNA
DIY method or use a kit

Question 3

DIY method of DNA extraction

Answer 3

Phenol-chloroform

Question 4

Steps of DNA extraction (phenol-chloroform)

Answer 4

1. Homogenize aka grind/suspend cells
2. Lysis (burst cells with detergent)
3. Acidification (digest cell components)
4. Extraction (phenol, chloroform)
   Slide 5

Question 5

Column based DNA extraction

Answer 5

Homogenize or suspend cells, burst cells (lysis), DNA binding to silica membrane, wash/let cell debris passed = pure DNA

Question 6

Two methods of DNA extraction

Answer 6

Phenol-chloroform
Column based (model organisms)

Question 7

Steps of mini-prep plasmid extraction

Answer 7

1. Resuspend cells
2. Lyse cells (high pH buffer)
3. Neutralize w buffer, anneal together for 2-3 mins
4. Separate cell debris
5. Steps for column-based
   Slide 7

Question 8

In mini-prep, why return pH to neutral and allow DNA to anneal back together?

Answer 8

Destroys genomic DNA, leaves plasmid for collection

Question 9

What is polymerase chain reaction

Answer 9

Put DNA polymerase in a test tube with everything in needs to synthesize DNA

Question 10

What does DNA polymerase require to synthesize DNA in vitro (PCR)? (5)

Answer 10

• 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)

Question 11

Slide 9

Answer 11

PCR

Question 12

Two key innovations in PCR

Answer 12

1. DNA polymerase from Thermus aquaticus (Taq) was discovered: stable at high temps
2. Development of automated thermocyclers

Question 13

What does gel electrophoresis do

Answer 13

Separate DNA molecules on the basis of their size

Question 14

How does gel electrophoresis work

Answer 14

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)

Question 15

What kind of gel is used in gel electrophoresis

Answer 15

Agarose or polyacrylamide

Question 16

Two ways of visualizing DNA in a gel

Answer 16

1. Ethidium bromide: intercalates between bases of nucleic acids, fluoresces red-orange in UV light
2. SYBR-green: forms complex w DNA that absorbs blue light, emits green

Question 17

Slides 13, 14

Answer 17

Visualizing DNA in gel

Question 18

What are restriction enzymes

Answer 18

Enzymes that cut dsDNA at specific sequences called restriction sites
Naturally occurring in bacteria

Question 19

What do bacteria use restriction enzymes for

Answer 19

RE (DNA endonucleases) used to destroy bacteriophage (virus that infect/destroy bacteria) chromosomes

Question 20

What do REs generate

Answer 20

Sticky ends

Question 21

Characteristics of the sequences REs cut at

Answer 21

4-8 bp sequences
Palindromic (read same forward as reverse)

Question 22

Why don’t restriction enzymes cut their own host DNA

Answer 22

Methylation hides the RE site from the enzyme, only added to host cell chromosome (but foreign DNA with seq will be cut)

Question 23

What is annealing

Answer 23

Single stranded nucleic acids naturally find complimentary nucleic acids to form double stranded nucleic acids

Question 24

What is recombinant DNA

Answer 24

Nucleic acids from different sources brought together through annealing phenomena

Question 25

2 main reasons we would want to use annealing in the lab

Answer 25

1. Connect or join any two strands of DNA from diff sources (recombinant DNA)
2. Find DNA among a sample that complements to a known piece of DNA (hybridization: allows us to identify unknown DNA)

Question 26

Slides 18, 19

Answer 26

Recombinant DNA, hybridization

Question 27

What is hybridization

Answer 27

Identification of unknown DNA through complementation

Question 28

Sanger sequencing uses

Answer 28

DNA extraction, PCR, gel electrophoresis

Question 29

How many PCR reactions in sanger sequencing?

Answer 29

Four

Question 30

Each PCR in sanger sequencing contains the normal components plus…

Answer 30

small amount of one of four dideoxynucleotides (stops DNA synthesis)

Question 31

Modern sanger sequencing uses…

Answer 31

Fluorescent labels on each ddNTP, reaction can be read in the same lane using a laser