cloning and genetic analysis

Question 1

what are restriction enzymes

Answer 1

prokaryotic defense system. recognize the harm and cut it out - they restrict what kind of DNA can exist in the host

Question 2

what are type II restriction endonucleases?

Answer 2

recognize inverted palindromic sequences in DNA

Question 3

when type II REs cut into palindromic sequences, they either leave…

Answer 3

sticky ends or blunt ends

Question 4

what does electrophoresis do?

Answer 4

separates DNA by their size

Question 5

what are the problems with cloning and how are they fixed?

Answer 5

1. not all cells will take up a plasmid - cells without plasmid will die in ampicillin because they don’t have the plasmid with the resistant gene
2. not all plasmids will take up the insert - insert will disrupt Lac Z gene and will not produce blue colour when interacting with X-gal

Question 6

what is a genomic library

Answer 6

a collection of DNA clones that contain copies of every DNA fragment in the whole genome inserted into a vector

Question 7

what is a DNA probe

Answer 7

single-stranded stretches of DNA of a known composition used to locate specific sequences in a genomic library (probe finds homologous sequences)

Question 8

true or false? probes do not have to be exact matches to the DNA they are locating

Answer 8

true. they will hybridize as long as their complementarity is more than 80%

Question 9

what is DNA sequencing?

Answer 9

after you have a PCR product or cloned product, you want to read the DNA sequence - provides highest resolution of a cloned DNA fragment

Question 10

what is the bottom line of Sanger dideoxy sequencing?

Answer 10

based on lack of 3’ OH group so that the chain is terminated

Question 11

what are some differences between PCR and sequencing?

Answer 11

PCR
- 2 RNA primers
- product is all the same
- double stranded product
Sequencing
- 1 RNA primer
- different sized products
- single stranded product