Recombinant DNA

Question 1

recombinant DNA

Answer 1

the isolation and manipulation of DNA sequences, usually involving combining nonconsecutive sequences

Question 2

what are the 2 types of enzymes that make DNA recombination possible

Answer 2

restriction endonuclease- cuts specific palandromic sequences, usually leaving sticky ends. usually palandrome sequences are 4/6/8 sequences long. they are named after the bacteria they are derived from

DNA ligase- reseals nicks in the backbone after insertion of DNA sequence

Question 3

What are the key characteristics of vectors?

Answer 3

1. separable from chromosomal DNA
2. restriction enzyme sites
3. OR and autonomous replication
4. able to tolerate sequence insertion
5. ability to be reinserted back into bacteria

Question 4

why are plasmids good vectors?

Answer 4

1. easily separable b/c of size
2. autonomous replication
3. tolerate insertion of ~10kb
4. can be engineered w/ restriction enzyme sites
5. encode selection factors

Question 5

what are alternatives to plasmids for vectors?

Answer 5

cosmids and bacterial artificial chromosomes- can encode bigger sequences but there are fewer per cell and they replicate much more slowly

Question 6

what is a genomic clone?

Answer 6

represents any part of the genome (including non coding material). therefore, can be taken from any cell. usually need cosmid or BAC b/c of larger protein sizes

Question 7

what is a library?

Answer 7

a genomic clone of the entire genome

Question 8

what is a cDNA clone and how is it different than genomic DNA

Answer 8

it represents a clone of only mRNA (has no introns). It matters where you get it from b/c tissues selectively express proteins. can be done with plasmids b/c they are generally smaller. you make a poly T tail as a primer, then RNase to degrade the original template

Question 9

describe how to run a southern blot

Answer 9

use restriction endonucleases to cut DNA. denature it and run it through a gel to separate by size. use capillary action to transfer the DNA onto a nitrocellulose sheet and soak it in solution with radioactive probe. visualize probe with x-rays- signal is autoradiography

Question 10

what is autoradiography

Answer 10

the signal at the end of a southern blot

Question 11

what is DNA polymorphism?

Answer 11

the genetic differences within individuals of a population, generally in non-coding regions