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Genomics > Enzymes And Restriction > Flashcards

Enzymes And Restriction Flashcards

1
Q

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

A

→ Insulin
→ Interferon- involved in antiviral defence

→ G-CSF- promotes formation of bone marrow.

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2
Q

What is G-CSF?

A

→ produced by infected cells and induces antiviral defense

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3
Q

What do nucleases do?

A

→ Degrade nucleic acids by hydrolysing phosphodiester bonds

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4
Q

What does ribonuclease degrade?

A

→ RNA

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5
Q

What does deoxyribonuclease degrade?

A

→ degrades DNA

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6
Q

What do exonucleases degrade?

A

→ degrades from the end of the molecule

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7
Q

What do endonucleases degrade?

A

→ degrades from the middle of the molecule

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8
Q

What is the purpose of restriction?

A

→ 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

→Recognize a specific sequence

→ cut a specific sequence

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10
Q

What sequence does ECOR1 recognise?

A

→ GAATTC

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11
Q

What are the characteristics of recognition sites?

A

→ 4-8 base pairs in length

→ they are palindromic

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12
Q

How often does a 4 base sequence occur?

A

→ every 265 bases

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13
Q

When does a 6 base recognition sequence occur?

A

→ every 4096 base pairs

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14
Q

What 2 nucleases produce an overhang?

A

→ EcoR1

→ Kpn1

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15
Q

What nuclease does not produce an overhang?

A

→Alu 1

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16
Q

How are DNA fragments separated in electrophoresis?

A

→ by charge

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17
Q

Where does the DNA move during electrophoresis and why?

A

→ DNA is negatively charged due to the phosphate groups

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

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18
Q

What is the relationship between migration and fragment size?

A

→ Smaller fragments run quicker

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19
Q

How do you find out the size of the fragment?

A

→ 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

→ it means you have two restriction sites

21
Q

What kind of a mutation occurs in sickle cell?

A

→ A single point mutation

→ Instead of GAG you get GTG

22
Q

What effect does the mutation have in sickle cell?

A

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

23
Q

What is the restriction site for the enzyme DDE1?

A

→ CTGAG

24
Q

How do you test for sickle cell using DDE1?

A

→ 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

25
Q

How can DNA molecules from different sourced joined together?

A

→ 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

26
Q

What does DNA polymerase do?

A

→ copies DNA based on a template

27
Q

Why do you use DNA polymerase?

A

→ PCR amplification
→ Generations of probes

→ blunt ending of DNA overhangs

28
Q

How does phosphatase work?

A

→ Hydrolyzes a phosphate group off its substrate

29
Q

What two types of phosphatases are used?

A

→ Calf intestinal phosphatase

→ Shrimp alkaline phosphatase

30
Q

Why do you use a phosphatase?

A

→ 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

31
Q

What does polynucleotide kinase do?

A

→ Converts phosphate from ATP to substrate

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

32
Q

Why would you use a polynucleotide kinase?

A

→ 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

33
Q

What is a probe?

A

→ fragment of ssDNA
→ 20-1000 bases in length

→ Complementary to the gene of interest

34
Q

What are reverse transcriptases isolated from and why?

A

→ isolated from RNA containing retroviruses- HIV

→Not found in eukaryotes

35
Q

What do reverse transcriptases do?

A

→ Synthesize DNA molecule complementary to a mRNA template using dNTPs

36
Q

What is needed to form a DNA copy from RNA?

A

→ a primer

37
Q

What is a random primer?

A

→ cDNA upto 700bp

→ covers all the length of the RNA molecules

38
Q

What is an oligo dT primer?

A

→ 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

39
Q

Why are specific primers designed for reverse transcriptase?

A

→ reverse transcriptase has a size limit

40
Q

What does palindromic mean?

A

reading in a certain direction on one strand is identical to the sequence in the same direction on the complementary strand

41
Q

Why do bacteria produce restriction enzymes?

A

→work as defence system for bacteria from bacteriophage.

→The restriction enzymes cleave the phage DNA​

42
Q

Why do restriction enzymes not cleave their own DNA?

A

because of methylation of bacteria DNA so not recognised by restriction ​enzymes

43
Q

How do phosphatases prevent resealing?

A

remove 5’-phosphate groups from DNA, RNA and both ribo and deoxy-ribonucleoside triphosphates

44
Q

Blunt ends or sticky ends, which has a lower efficacy ligation?

A

blunt ends

45
Q

In SCA, what restriction site is lost?

A

DdeI site (5’CTNAG3’)

Genomics (21 decks)

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