Topic 6: Molecular Genetics Part 2

Question 1

Blotting techniques use…

Answer 1

Restriction enzymes, gel electrophoresis, hybridization

Question 2

Why label DNA in blotting? How?

Answer 2

Labeled DNA can be used as a probe for specific gene sequences
Label with nucleotides labeled with radioactive 32P, visualized using x-ray film

Question 3

Goal of blotting

Answer 3

Find a unique sequence in a mixture of DNA/RNA separated in a gel

Question 4

Slide 5

Answer 4

Southern blot

Question 5

What is a southern blot test

Answer 5

DNA or RNA probe on DNA sample
Genome to genome comparisons
Restriction fragment length polymorphs (RFLP) mapping or genotyping

Question 6

What is a northern blot

Answer 6

RNA/DNA probe on RNA sample
Determine gene expression (mRNA only present if your gene of interest is present)
Looking for expression in different tissues or under diff treatments

Question 7

What is a western blot test

Answer 7

Antibody probe on a protein sample (not based on complementary hybridization)
Look for specific protein in unknown

Question 8

Slide 7***

Answer 8

Reading southern blots, pic on phone

Question 9

What is in situ hybridization

Answer 9

Hybridization that is done within the organism (instead of in vitro)
Can be done with DNA, RNA or protein

Question 10

Cloning uses..

Answer 10

Restriction enzymes, recombinant DNA technology, mini-prep

Question 11

What are clones

Answer 11

Identical organisms, cells or molecules (molecular cloning = DNA) derived from a single ancestor

Question 12

Why clone

Answer 12

Get a lot of fragments of DNA of interest
Keep it stably replicating using bacterial host (E coli)

Question 13

Four steps of cloning (slide 11)

Answer 13

1. Extract a d purify (target DNA and plasmid vector)
2. Digest target with RE
3. Digest vector with same RE
4. Ligation (mix vector + target together with ligase)
5. Recombinant DNA is formed

Question 14

Characteristic of vectors for cloning

Answer 14

• must be able to replicate itself and DNA segment it carries (ORI)
• several restriction sites present only once in the vector
• selectable markers (antibiotic resistance genes or reporter genes)
• easy to recover from the host cell (via mini-prep)

Question 15

How do we find bacteria that have our plasmid with the recombinant (cloning)

Answer 15

Ampicillin resistance gene is in pUC19, host cell will be able to grow on ampicillin plates (if it has plasmid pUC19)

Question 16

Slide 13

Answer 16

pUC19

Question 17

Slide 14

Answer 17

?

Question 18

What is transformation

Answer 18

Process of putting foreign DNA into any organism (vector-> bacteria)

Question 19

What is a competent cell

Answer 19

Bacteria cells that are primed to take up plasmids

Question 20

Fifth and sixth steps of cloning

Answer 20

5. Transformation of plasmid DNA into bacterial host
6. Selection of transformed bacteria

Question 21

How do we select for transformed bacteria (with the recombinant plasmid)

Answer 21

Grow on plate with ampicillin and X-gal
1. Bacteria that don’t have plasmid don’t grow (no ampicillin resistance)
2. bacteria w/ empty plasmid - no foreign DNA (grows blue)
3. Bacteria with recombinant plasmid (grow white/colourless)

Question 22

Slides 17, 18, 19

Answer 22

Ti plasmid in Agrobacterium tumefaciens

Question 23

DNA fingerprinting uses…

Answer 23

DNA extraction, PCR, automated sequencing

Question 24

What are microsatellites

Answer 24

Short tandem repeats (STRs), variable number of copies of repeat sequences possessed by many organisms
Detected by PCR

Question 25

Slides 20-> 25

Answer 25

DNA fingerprinting