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SGUL - Genomics > Enzyme And Restriction Mapping > Flashcards

Enzyme And Restriction Mapping Flashcards

1
Q

What are Nucleases?

A

These are enzymes that degrade the nucleic acids by hydrolysing phosphodiester bonds

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2
Q

What are the two types of nucleases?

A
  • Ribonuclease (RNAase): Degrade RNA
  • Deoxyribonuclease (DNase): Degrade DNA
    ExoNuclease: Degrade from the end of molecule
    Endoculease: Cleave within the nucleotide chain
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3
Q

What are restriction enzymes?

A

Restriction: Limit the transfer of nucleic acids from infecting phases into bacteria. May have different enzymes from bacteria

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4
Q

What do restriction enzymes do?

A
  • Recognise a specific sequence
  • Cut that sequence
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5
Q

View diagrams

A

Different restriction enzymes recognise different specific DNA sequences

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6
Q

In Theory, where does a 4 base recognition sequence occur?
Also known as Short Tandem Repeats

A

Every 256 bases. (4x4x4x4)

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7
Q

In Theory, where does a 6 base recognition sequence occur?
Also known as Short Tandem Repeats

A

Every 4096 bases (4x4x4x4x4x4)

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8
Q

What is an overhang and what is it produced by?

A
  • Some nucleases produce overhangs
  • This is where the DNA sequence is cut between the 5’ and 3’ UTRs leaving a gap between two bases
  • This cut then causes another cut of 4 bases on the corresponding sequence causing it to overhang
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9
Q

What are the two types of cuts caused by nucleases?

A

Overhangs and blunt ends

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10
Q

What are Restriction Enzymes useful for?

A
  • Cloning
  • Molecular diagnostics
  • Characterisation of plasmids
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11
Q

What type of enzyme could be used to bring two DNA molecules together?

A

DNA ligaments is used to create a phosphodiester bond between the human DNA and bacterial DNA to create a recombinant DNA molecule

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12
Q

What was restriction enzymes first used to diagnose in Molecular Diagnostics?

A

Sickle cell Anaemia

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13
Q

How can Restriction enzyme sites be destroyed?

A

By single nucleotide changes which changes the structure of the site

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14
Q

What are the Restriction maps uses of restriction enzymes?

A
  • Map of restriction sites within a DNA molecule
  • Crude way of mapping an unknown molecule
  • A Useful way of describing plasmids
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15
Q

What are Plasmid maps?

A

Essentially tells you where restriction enzymes digests the plasmid

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16
Q

Watch Lecture to get an idea of Mapping

A
17
Q

What other enzymes are used when creating recombinant DNA?

A
  • DNA ligase
  • DNA polymerase
  • Phosphatases
  • Polynucleotide kinase
  • Reverse transcriptase
18
Q

What is the function of DNA ligase?

A
  • Repairs nicks in the phosphodiester backbone
  • Creates new phosphodiester bonds between the phosphate group and hydroxyl group between the 5’ and 3’
19
Q

What is the function of DNA polymerase?

A
  • Carry out DNA synthesis in the 5’ to 3’ direction
  • To do this, they need a primer
20
Q

What are the other uses of DNA polymerase?

A
  • PCR amplification
  • Generation of probes
  • Blunt ending of DNA overhangs
21
Q

What is the function of Phosphatase?

A

Hydrolyses the phosphate group off of its substrates
- Cald intestinal alkaline Phosphatase were previously used
- Shrimp Alkaline Phosphatase are now used

22
Q

Why would you use a Phosphatase?

A

To prevent cut plasmids from resealing

23
Q

What is the function of polynucleotide Kinase?

A

Incorporates a phosphate group from ATP to the substance
- reaction: ATP + Subtrate -> ADP + phosphorylated substrate
- Polynucleotide kinase adds phosphate to 5’ hydroxyl group of DNA or RNA

24
Q

Why would we use a Polynucleotide kinase?

A
  • To phosphorylase chemically synthesised DNA so that it can be lighted to another form
  • To sensitively label DNA so that it can be traced using
  • Radioactivity labelled ATP or Fluorescently lebelled ATP
25
Q

Describe Probes

A
  • Fragments of ssDNA (or RNA)
  • 20 - 1000 bases in length
  • Complementary to the gene of interest
26
Q

Describe Reverse Transcriptase

A
  • Uses RNA as a template
  • RNA dependent DNA polymerase
  • Isolated from RNA containing retroviruses
  • Synthesizes a DNA molecule complementary to a mRNA template using dNTPs
  • Needs a primer aswell
27
Q

What type of primers could you use for Reverse Transcription?

A
  • Random Primers: cDNAs upto 700 bp but will cover all the length of all of the RNA molecules
  • Oligo (dT) primer: Useful for cloning cDNAs and cDNA libraries but some might not be full length
28
Q

What is the issue with using an oligo primer?

A

Sometimes the primer does not reach the end of the rna molecule which causes problems trying to clone the full gene

29
Q

How is this issue overcome?

A

This issue is overcome by using gene specific primers. This makes it more likely that you would reach the end of the RNA molecule

SGUL - Genomics (21 decks)

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