Manipulating and Cloning DNA

What are restriction nucleases, and what did their discovery show about bacteria?

Bacterial enzymes that cut DNA at specific 4–8 bp sequences. They showed bacteria destroy foreign DNA by cutting (restricting) it; each bacterium makes different ones.

What is the difference between sticky and blunt ends?

Sticky = single-stranded overhangs (EcoRI, HindIII, NotI). Blunt = cut straight across (AluI, SmaI, HaeIII).

On an agarose gel, where do large fragments end up, why does DNA move toward the + electrode, and how is it seen?

Large fragments migrate slowly and stay near the top. DNA is negatively charged, so it moves toward +. Ethidium bromide intercalates and fluoresces under UV; a ladder gives fragment sizes.

How many fragments do n cuts give for linear vs circular DNA?

Linear: n + 1. Circular (plasmid): n (one cut just opens the circle). With multiple enzymes, add up all cut sites first.

A linear piece has sites in the order EcoRI, HindIII, HindIII, EcoRI. How many fragments with both enzymes?

5 (4 cuts). EcoRI alone = 3; HindIII alone = 3.

A 4000 bp piece has segments (left to right) of 1000, EcoRI site, 2000, HindIII site, 800, HindIII site, 200. What fragments are in the HindIII lane?

3000, 800, and 200 bp. (EcoRI lane: 3000, 1000. Both enzymes: 2000, 1000, 800, 200.)

Which enzymes leave 3′ sticky ends?

PstI (CTGCA/G), KpnI, PacI. (5′ sticky: EcoRI, BamHI, Sau3A, NotI. Blunt: SmaI, AluI.)

How do you denature and renature DNA, and what does hybridization let you do?

High temperature or high pH denatures DNA into single strands; slow cooling or lower pH renatures it. Hybridization lets a labeled probe find its complementary sequence.

Which techniques detect DNA and which detect RNA?

DNA: Southern blotting, CGH. RNA: Northern blotting, in situ hybridization, microarrays.

How does hybridization identify a person with one normal and one sickle-cell β-globin gene?

Both the normal-gene probe and the sickle-cell probe bind. (Sickle differs by one base, GAG → GTG, Glu → Val. Normal probe only = 2 normal genes; sickle probe only = 2 sickle genes.)

What does DNA ligase do?

Joins DNA fragments covalently to make recombinant DNA; uses ATP. Fragments cut by different enzymes can be joined after DNA polymerase + dNTPs fill in the staggered ends.

What must a plasmid vector carry?

A promoter, an origin of replication, and antibiotic selection markers (it is a circular carrier cut with the same enzyme as the insert).

What are the four steps of DNA cloning in bacteria?

1) Cut DNA with restriction nucleases. 2) Join with ligase to make recombinant DNA. 3) Insert into a plasmid vector. 4) Introduce into bacteria, grow the culture, and purify plasmid copies.

How would you overexpress a protein?

Clone the gene into an expression vector with a strong promoter so cells make overexpressed mRNA and protein.

How would you see where a protein sits in the cell?

Fuse the gene to GFP (a jellyfish protein) so the chimeric protein glows, then look for fluorescence. (Examples: GFP/RFP-tagged oncogene in ovarian cancer cells; GFP mouse.)

What are the ways to study a gene's function?

Overexpress it, delete or knock it down, mutate it (site-directed mutagenesis), then measure the functional response.