Analytical tools

Question 1

Restriction enzymes

Answer 1

Endonucleases that cleave alpha-phosphodiester bond in DNA
Type 1: cleaves at 1000bp around specific sequence (contains methylase & req ATP)
Type 2: recognizes pallindromes 6-8bp; cuts assymetrically
Type 3: cleaves 10bp around specific sequence (contains methylase & req ATP)

*bacteria protect their own DNA via methylation of recognition sequences

Question 2

Vector cloning

Answer 2

1. RE cut plasmid vector & chromosomal DNA
2. DNA ligase binds sticky ends
3. Transformation into host cell via heat shock & CaCl
4. Amplification/propagation –> many copies
5. Selection: antibiotic resistance to select for transformed cells

Question 3

Vector cloning: pBR322 example

Answer 3

1. EcoR1 (sticky) and PvuII/pst1 (blunt) RE
2. Transformation w/ tetracycline (colonies contain plasmid)
3. Then positively select by transferring colonies onto agar w/ tetracycline & ampicillin
4. Colonies no longer present in test plate, contain recombinant plasmids (since insertion of chromosome DNA disrupts amp resistance)

Question 4

Lamda bacteriophage cloning

Answer 4

1. 40-50kb bacterial virus that can incorporate larger genes
2. Contains genes for replication & packaging that are essential
3. insert DNA w/ RE and ligase into fill DNA region
4. Fragments that lack essential DNA will be too small to be packaged
5. Lamda bacteriophage now contain foreign DNA and can infect host cell

Question 5

BAC vector

Answer 5

1. Contains: ori, chloramphenicol resistance (cmR), par genes (limit replication to single copy), and lacZ in cloning site
2. Use ELECTROPORATION (high voltage current opens membrane) for transformation
3. Selection on agar w/ chloramphenicol, then w/ substrate for beta-galactosidase
4. Recombinant colonies appear white b/c they are unable to metabolize gal since lacZ interrupted
   * Colonies with normal plasmid –> blue

Question 6

Polymerase chain reaction

Answer 6

1. heat to separate strands
2. When cool, add synthetic oligonucleotide primers
3. add taq DNA pol (thermostable) –> syn
4. Continue amplification for several rounds
   * after 25 cycles: amplify target sequence 10^6 fold
   * can then be cloned into plasmid vector

Question 7

Gel electrophoresis

Answer 7

1. Agarose (DNA/RNA) or polyacrylamide (protein) gel separates sample based on size
2. Ethidium bromide intercalates into CG nucleic acids –> fluoresces under UV light

Question 8

SDS PAGE

Answer 8

SDS detergent: Denatures and coats with negative charge

Used for proteins

Question 9

Southern blot & Northern blot

Answer 9

southern: DNA northern: RNA
*RNA denatured by formaldehyde
1. Transferred/blotted onto nitrocellulose or nylon membrane
2. hybridized with radioactive probe; excess probe washed off
3. Visualized w/ autoradiography
*quantitative and can detect changes in size
insertion/deletion, amplification, translocation
*DNA: restriction length polymorphisms for paternity and forensics

Question 10

Western blot

Answer 10

For proteins

1. electrotransferred to a nitrocellulose membrane
2. Incubated with radioactively tagged antibodies

Question 11

DNA Microarray

Answer 11

1. Isolate mRNA from two cells from different stages of development
2. Convert to cDNA via reverse transcriptase and fluorescently labeled dNTPs –> probes
3. Add to microarray that contain ss genes –> hybridization
4. Each colored spot represents expressed gene in the cells; mix= expressed equally

Question 12

Sanger method: DNA sequencing

Answer 12

1. Primer is annealed to template DNA (SS or denatured ds) —> DNA synthesis
2. Add 3 normal dNTPs and last one with a mix of ddNTP and dNTPs
3. Result in fragments of various lengths
4. Can separate by size on gel to sequence
   fluorescently
   *If ddNTPs are labeled, can use a computer to detect and generate sequence