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πšπšŽπš—πš˜πš–πš’πšŒπšœ > Enzymes and Restriction Mapping > Flashcards

Enzymes and Restriction Mapping Flashcards

1
Q

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

A

β†’ Insulin
β†’ Interferon
β†’ G-CSF

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2
Q

What is G-CSF?

A

β†’ produced by infected cells and induces antiviral defense

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3
Q

What do nucleases do?

A

β†’ Degrade nucleic acids by hydrolysing phosphodiester bonds

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4
Q

What does ribonuclease degrade?

A

β†’ RNA

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5
Q

What does deoxyribonuclease degrade?

A

β†’ degrades DNA

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6
Q

What do exonucleases degrade?

A

β†’ degrades from the end of the molecule

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7
Q

What do endonucleases degrade?

A

β†’ degrades from the middle of the molecule

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8
Q

What is the purpose of restriction?

A

β†’ to limit the transfer of nucleic acids from infecting phages into bacteria

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9
Q

What 2 things do restriction endonucleases do?

A

β†’Recognize a specific sequence

β†’ cut a specific sequence

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10
Q

What sequence does ECOR1 recognise?

A

β†’ GAATTC

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11
Q

What are the characteristics of recognition sites?

A

β†’ 4-8 base pairs in length

β†’ they are palindromic

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12
Q

How often does a 4 base sequence occur?

A

β†’ every 265 bases

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13
Q

When does a 6 base recognition sequence occur?

A

β†’ every 4096 base pairs

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14
Q

What 2 nucleases produce an overhang?

A

β†’ EcoR1

β†’ Kpn1

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15
Q

What nuclease does not produce an overhang?

A

β†’Alu 1

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16
Q

How are DNA fragments separated in electrophoresis?

A

β†’ by charge

17
Q

Where does the DNA move during electrophoresis and why?

A

β†’ DNA is negatively charged due to the phosphate groups

β†’ when a current is applied the DNA moves to the +ve pole

18
Q

What is the relationship between migration and fragment size?

A

β†’ Smaller fragments run quicker

19
Q

How do you find out the size of the fragment?

A

β†’ You compare the digested fragments to the standard ones to compare the length

20
Q

What does having two bands in a linear fragment mean?

A

β†’ it means you have two restriction sites

21
Q

What kind of a mutation occurs in sickle cell?

A

β†’ A single point mutation

β†’ Instead of GAG you get GTG

22
Q

What effect does the mutation have in sickle cell?

A

β†’ induces charges in the beta globin gene which makes it defective

23
Q

What is the restriction site for the enzyme DDE1?

A

β†’ CTGAG

24
Q

How do you test for sickle cell using DDE1?

A

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

25
Q

How can DNA molecules from different sourced joined together?

A

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

26
Q

What does DNA polymerase do?

A

β†’ copies DNA based on a template

27
Q

Why do you use DNA polymerase?

A

β†’ PCR amplification
β†’ Generations of probes
β†’ blunt ending of DNA overhangs

28
Q

How does phosphatase work?

A

β†’ Hydrolyzes a phosphate group off its substrate

29
Q

What two types of phosphatases are used?

A

β†’ Calf intestinal phosphatase

β†’ Shrimp alkaline phosphatase

30
Q

Why do you use a phosphatase?

A

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

31
Q

What does polynucleotide kinase do?

A

β†’ Converts phosphate from ATP to substrate

β†’ Adds phosphate to the 5’ hydroxyl group of DNA or RNA

32
Q

Why would you use a polynucleotide kinase?

A

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

33
Q

What is a probe?

A

β†’ fragment of ssDNA
β†’ 20-1000 bases in length
β†’ Complementary to the gene of interest

34
Q

What are reverse transcriptases isolated from and why?

A

β†’ isolated from RNA containing retroviruses

β†’Not found in eukaryotes

35
Q

What do reverse transcriptases do?

A

β†’ Synthesize DNA molecule complementary to a mRNA template using dNTPs

36
Q

What is needed to form a DNA copy from RNA?

A

β†’ a primer

37
Q

What is a random primer?

A

β†’ cDNA upto 700bp

β†’ covers all the length of the RNA molecules

38
Q

What is an oligo dT primer?

A

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

39
Q

Why are specific primers designed for reverse transcriptase?

A

β†’ reverse transcriptase has a size limit

πšπšŽπš—πš˜πš–πš’πšŒπšœ (19 decks)

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