ch 10

Question 1

restriction enzymes?

Answer 1

recognize a specific sequence of bases anywhere within the genome and create a double stranded break

Question 2

palindromic symmetry

Answer 2

a sequence that goes 1 way 5 to 3 and it is the exact same sequence just in the oppo

Question 3

what are the 2 assumptions with fragments?

Answer 3

Each of the four bases occur in equal proportion (25% each)
Bases are distributed randomly in the DNA sequence

Question 4

what are the three types of cuts that use palindromic symmetry?

Answer 4

Blunt : straight down the middle
Stickyends : they will clip at the edges leaving an overhang either at 5’ (sticky 5’ end) or at 3’ (sticky 3’ end)

Question 5

what does a longer recognition site mean?

Answer 5

longer the fragment will be less likely to be found in the genome

Question 6

formula for calculating average size fragment

Answer 6

4^n

Question 7

how can you find how many fragments will be in the genome?

Answer 7

Base pairs/ average size of the fragment= how many times they are found

Question 8

what is gel electrophoresis ? and how can fragments move in them?

Answer 8

-Movement of charged particles through an electric field where DNA is then seperated by size -
smaller runs the fastest

Question 9

what is molecular cloning

Answer 9

a way to isolate the fragment of interest and make copies of them

Question 10

how does molecular cloning work?

Answer 10

Step 1:DNA fragment is inserted into the DNA plasmid by using a restriction enzyme which will allow the sticky end overhangs to hybridize
Step 2: Plasmid and the DNA fragment are transferred into bacterial cells for mass production

Question 11

what affect does heat or electircaly shocking the DNA fragments?

Answer 11

This temporarily increases the permeability of bacterial cell membrane allowing for DNA to enter briefly

Question 12

How can you be sure that the 2 sticky ends won’t just connect back together?

Answer 12

Cut with 2 enzymes that make 2 different sticky ends
Same compatible ends so the insert can glue them back together

Question 13

What enzymes glues everything back together?

Answer 13

DNA ligase

Question 14

what are the three properties of plasmids?

Answer 14

-polylinker (short DNA sequence with many different restriction sites)
-origin of replication
-selectable marker (gene of interest and if the plasmid contains this it will survive if not dead)

Question 15

genomic libraries

Answer 15

a long lived collection of cellular clones that contains copies of every sequence in the whole genome inserted into a suitable vector

Question 16

What happens if the genome is cut with a single restriction enzyme until completion?

Answer 16

Tandem fragments are created - you would not be able to tell the order of the fragments

Question 17

how can you solve the issue of tandem repeats?

Answer 17

Partial digestion: only a fraction of restriction sites - creating overlapping fragments of different sizes
- need decreasing enzyme concentration and decreasing reaction time

Question 18

What do you need for DNA replication?

Answer 18

-DNA polymerase (tells where to start making the fragment)
-template:
-Deoxyribonucleotide triphosphate ( d(any 4 base pair)tp )
-Primers

Question 19

How do we unwind and separate the DNA strand?

Answer 19

Heat breaks the H-bonds

Question 20

how can you make fragments?

Answer 20

-dentauring the DNA will let the primers seperate
-they will pair up with complementary regions on the single strand of DNA
-DNA pol uses special nucleotides (dideoxyribose- no OH groups dd?TP ) once added stops the growth of DNA
-can graph and each peak tells you the base

Question 21

Whole genome shot gun sequencing? issue? solution?

Answer 21

creates Continuous base-pair sequencing
-issue is that there are Repetitive sequences in the genome might cause incorrect assembly and repeats longer than the fragment
-paired-end sequencing solves the issue by using BAC (longer fragments)