enzyme and restriction mapping

Question 1

What are restriction enzymes?

Answer 1

• an enzyme that has the ability to cleaving DNA molecules at near or specific sequence of bases.

Question 2

Where are restriction enzymes produced?

Answer 2

• produced in some bacteria cells.

- macrophages attack bacteria and incorporate their genome but restriction enzymes cleave this out - defense mechanism.

Question 3

Why do restriction enzymes only cleave of macrophages genome and not their own?

Answer 3

• restriction enzymes dont cleave off bacteria’s own DNA bc when DNA in bacteria is replicated its methylated
• CH3 acts as a tag so restriction enzymes can recognise their own DNA.

Question 4

Define restriction.

Answer 4

• limit transfer of nucleic acids from infecting phages into bacteria.
• History : this was observed by scientists when they saw less viral plaque forming in E. coli strain K , there must be something “restricting” growth and later named it restriction enzyme.

Question 5

Which type of restriction enzyme cleaves specifically within restriction sites?

Answer 5

• type 2 => isolated from hemophilus influenza bacteria (called HIND 2)
• type 1 and 3 cleave randomly

Question 6

Define recombinant DNA?

Answer 6

• the plasmid that is made from ligating multiple genes is known as recombinant DNA.
• one part comes from human DNA and one part comes from bacterial DNA.

Question 7

What are some genetic engineering uses of recombinant protein?

Answer 7

=> insulin
=> interferon : protein used for antiviral infection
=> G-CSF : promotes formation of bone marrow, chemotherapy degrades bone marrow in cancer patients.

Transgenic organisms (Mice) are used to model disease.

Question 8

What are 2 types of nucleases?

Answer 8

1. ribonucleases (RNase)

2. deoxyribonuclease (DNase)

Question 9

What are the 2 subtypes pf DNase?

Answer 9

=> exonucleases : degrade from the ends of the nucleotide chain.
=> endonucleases : degrade from within nucleotide chain

Question 10

What is the function of nucleases?

Answer 10

• degrade nucleic acids by hydrolysing phosphodiester bonds.

Question 11

What is a palindromic sequence?

Answer 11

• reads the same in the opposite strand.

Question 12

Where does the restriction enzyme EcoR1 cleave?

Answer 12

• GAATTC (6 bases)

- cleaves at G forming a sticky overhang

Question 13

how many times does a 4 base and a 6 base recognition sequence occur?

Answer 13

• 4 bases (4^4 = 256 bases)

- 6 bases ( 4^6 = 4096 bases)

Question 14

Which nuclease produces a 3’ sticky end overhang?

Answer 14

• Kpn1

Question 15

Which nuclease produces a 5’ sticky end overhang?

Answer 15

• EcoR1

Question 16

which nuclease produces a blunt end overhang?

Answer 16

• Alu1

- 4 base blunt end (256 bases)

Question 17

What is restriction mapping?

Answer 17

a restriction map is a map of known restriction sites within a sequence of DNA.
useful way of describing plasmids.

Question 18

How many base plasmid is pDTL3?

Answer 18

• 13 kilo base plasmid (13kb)

Question 19

How many fragments result from Bam HI and what bases are they?

Answer 19

• 2 fragments result
• 6kb and 7kb
• add upto 13 kb

Question 20

how many fragments result from EcoRI digesting the plasmid?

Answer 20

• 1 fragment

- 1 cut