Enzymes and Restriction Mapping

Question 1

What are 3 recombinant proteins that are made using genetic engineering?

Answer 1

→ Insulin
→ Interferon
→ G-CSF

Question 2

What is G-CSF?

Answer 2

→ produced by infected cells and induces antiviral defense

Question 3

What do nucleases do?

Answer 3

→ Degrade nucleic acids by hydrolysing phosphodiester bonds

Question 4

What does ribonuclease degrade?

Answer 4

→ RNA

Question 5

What does deoxyribonuclease degrade?

Answer 5

→ degrades DNA

Question 6

What do exonucleases degrade?

Answer 6

→ degrades from the end of the molecule

Question 7

What do endonucleases degrade?

Answer 7

→ degrades from the middle of the molecule

Question 8

What is the purpose of restriction?

Answer 8

→ to limit the transfer of nucleic acids from infecting phages into bacteria

Question 9

What 2 things do restriction endonucleases do?

Answer 9

→Recognize a specific sequence

→ cut a specific sequence

Question 10

What sequence does ECOR1 recognise?

Answer 10

→ GAATTC

Question 11

What are the characteristics of recognition sites?

Answer 11

→ 4-8 base pairs in length

→ they are palindromic

Question 12

How often does a 4 base sequence occur?

Answer 12

→ every 265 bases

Question 13

When does a 6 base recognition sequence occur?

Answer 13

→ every 4096 base pairs

Question 14

What 2 nucleases produce an overhang?

Answer 14

→ EcoR1

→ Kpn1

Question 15

What nuclease does not produce an overhang?

Answer 15

→Alu 1

Question 16

How are DNA fragments separated in electrophoresis?

Answer 16

→ by charge

Question 17

Where does the DNA move during electrophoresis and why?

Answer 17

→ DNA is negatively charged due to the phosphate groups

→ when a current is applied the DNA moves to the +ve pole

Question 18

What is the relationship between migration and fragment size?

Answer 18

→ Smaller fragments run quicker

Question 19

How do you find out the size of the fragment?

Answer 19

→ You compare the digested fragments to the standard ones to compare the length

Question 20

What does having two bands in a linear fragment mean?

Answer 20

→ it means you have two restriction sites

Question 21

What kind of a mutation occurs in sickle cell?

Answer 21

→ A single point mutation

→ Instead of GAG you get GTG

Question 22

What effect does the mutation have in sickle cell?

Answer 22

→ induces charges in the beta globin gene which makes it defective

Question 23

What is the restriction site for the enzyme DDE1?

Answer 23

→ CTGAG

Question 24

How do you test for sickle cell using DDE1?

Answer 24

→ purify DNA
→ Fragment of 450 bp
→ digest the PCR with DDE1
→ if there is normal beta globin - 2 restriction sites and 3 fragments
→ if there is sickle cell beta globin - 1 restriction site and 2 fragments

Question 25

How can DNA molecules from different sourced joined together?

Answer 25

→ ECOR1 overhangs are compatible → Two fragments are mixed with DNA ligase and a recombinant molecule is produced → DNA ligase makes covalent phosphodiester bonds between DNA fragments

Question 26

What does DNA polymerase do?

Answer 26

→ copies DNA based on a template

Question 27

Why do you use DNA polymerase?

Answer 27

→ PCR amplification → Generations of probes → blunt ending of DNA overhangs

Question 28

How does phosphatase work?

Answer 28

→ Hydrolyzes a phosphate group off its substrate

Question 29

What two types of phosphatases are used?

Answer 29

→ Calf intestinal phosphatase | → Shrimp alkaline phosphatase

Question 30

Why do you use a phosphatase?

Answer 30

→ To prevent cut plasmids from resealing → If the phosphate group in the plasmid isn't removed it can reseal → If it is removed then it cannot be resealed alone because DNA ligase needs a phosphatase group to work on one single size

Question 31

What does polynucleotide kinase do?

Answer 31

→ Converts phosphate from ATP to substrate | → Adds phosphate to the 5' hydroxyl group of DNA or RNA

Question 32

Why would you use a polynucleotide kinase?

Answer 32

→ To phosphorylate chemically synthesized DNA so that it can be ligated to another fragment → To sensitively label DNA so that is can be traced using radioactive or fluorescently labelled ATP

Question 33

What is a probe?

Answer 33

→ fragment of ssDNA → 20-1000 bases in length → Complementary to the gene of interest

Question 34

What are reverse transcriptases isolated from and why?

Answer 34

→ isolated from RNA containing retroviruses | →Not found in eukaryotes

Question 35

What do reverse transcriptases do?

Answer 35

→ Synthesize DNA molecule complementary to a mRNA template using dNTPs

Question 36

What is needed to form a DNA copy from RNA?

Answer 36

→ a primer

Question 37

What is a random primer?

Answer 37

→ cDNA upto 700bp | → covers all the length of the RNA molecules

Question 38

What is an oligo dT primer?

Answer 38

→ useful for cloning cDNAs and cDNA libraries but some might not be full length → a TTT sequence is used because genes that code for proteins are polyadenylated

Question 39

Why are specific primers designed for reverse transcriptase?

Answer 39

→ reverse transcriptase has a size limit