7. ENZYME & RESTRICTION MAPPING

Question 1

What are some of the application of Genetic engineering?

Answer 1

Restriction enzymes can be used to:

1. Produce recombinant proteins such as insulin, interferon, G-CSF
2. Produce transgenic organisms & plants

Question 2

What do nucleases do?

Answer 2

Nucleases degrade nucleic acids by hydrolyzing the phosphodiester bonds between the nucleotides

Question 3

What are the two types of nucleases?

Answer 3

1. Ribonucleases - degrade RNA
2. Deoxyribonucleases - degrade DNA
   * There are two types deoxyribonucleases:
   - - >EXONUCLEASES - cleave the end of the sequence
   - -> ENDONUCLEASES - cleave within the sequence

Question 4

*Where do restriction enzymes/endonucleases originate from?

Answer 4

• Restriction enzymes are originally found/produced in bacteria. Different types of bacteria will produce different restriction enzymes
• Infecting phages or bacteriophages inject their nucleic acids into bacteria, so bacteria have restriction enzymes to counteract this.
  The bacteria cleave the nucleic acids, limiting their transfer
• Restriction enzymes in bacteria only cleave foreign DNA not their own because bacteria’s DNA is methylated

Question 5

What do restriction enzymes do?

Answer 5

1. Recognise specific sequences

2. Cut/cleave the sequence by hydrolysing phosphodiester bonds between the nucleotides

Question 6

**What are recognition sites?

Answer 6

• Recognition sites are 4-8 bases in length, depending on the restriction enzyme
• They are palindromic meaning they can be read in either direction (5’3’ or 3’5’)
• Recognition sequence of 4 bases = 4^4 = 256

Question 7

What are examples of restriction enzymes?

Answer 7

• EcoRI
• BamHI
• HindII

Question 8

What are two outcomes of restriction enzyme cleavage?

Answer 8

1. Blunt ends

2. Overhang - produced when phosphodiester bonds are cleaved

Question 9

Give examples of nucleases which produce an overhang & blunt ends?

Answer 9

• EcoRI produces a 5’ overhang
• KpnI leaves a 3’ overhang
• AluI produces blunt ends

Question 10

What are three uses of restriction enzymes?

Answer 10

1. Cloning
2. Molecular Diagnosis
3. Inserting genes into plasmid/characterisation of plasmids

Question 11

How can single nucleotide changes like SCD affect recognition sites?

Answer 11

• A single nucleotide change can change the recognition site
• E.g The restriction enzyme DdeI recognises the sequence CTGAG.
• BUT… in SCD, a single base substitution changes GAGA to GTG so the sequence now reads CTGTG.
• The recognition site is lost & DdeI cannot recognise the sequence
• This will show up as a missing site on PCR

Question 12

What are restriction maps?

Answer 12

• Restriction maps are a visual representation showing the restriction sites for enzymes

Question 13

What five enzymes are involved in forming recombinant DNA?

Answer 13

1. DNA LIGASE
2. DNA POLYMERASE
3. PHOSPHATASE
4. POLYNUCLEOTIDE KINASE
5. REVERSE TRANSCRIPTASE

Question 14

What does DNA ligase do in genetic engineering?

Answer 14

• DNA LIGASE is a ligating enzyme which joins the two fragments of DNA together.
• It makes new phosphodiester bonds between the cleaved sites

Question 15

Why is DNA Polymerase used in genetic engineering?

Answer 15

• DNA Polymerase synthesises DNA from 5’ to 3’.
• DNA polymerase is needed for the blunt ending of the overhangs
• The overhang acts as a primer, so when DNA polymerase is added with nucleotides it can extend the overhang to produce a blunt end (double stranded)

Question 16

What do phosphatases do in genetic engineering & why are they used?

Answer 16

• Phosphatases remove phosphates by hydrolyzing the phosphate group of a substrate
• Calf intestinal alkaline substrate or shrimp alkaline substrate can be used
• Phosphatases are needed to prevent cut plasmids from reannealing

Question 17

What does a polynucleotide kinase do in genetic engineering & why are they used?

Answer 17

• Polynucleotide kinases add a phosphate to the 5’ hydroxyl of the DNA or RNA using ATP
• Chemically synthesised DNA isn’t phosphorylated, so it needs to be phosphorylated before it can be ligated & cloned
• Polynucleotide kinase also helps label DNA by using radioactive or fluorescent ATP

Question 18

What does reverse transcriptase do in genetic engineering?

Answer 18

• Reverse transcriptase is a RNA dependent DNA polymerase which is isolated from retroviruses containing RNA
• It synthesises cDNA from the mRNA template using dNTPs

Question 19

What three primers can be used in reverse transcription?

Answer 19

1. Random
2. Oligo (dT)
3. Gene specific

Question 20

*How is a random primer used in reverse transcription?

Answer 20

• A random primer is a 6 nucleotides long with any composition, but it binds to the sequence with perfect complementarity
• cDNA will be made in fragments
• But some very long mRNAs won’t be transcribed

Question 21

How is an Oligo(dT) primer used in reverse transcription?

Answer 21

• An Oligo (dT) primer is a string of deoxythymidic bases (poly T tail). This poly T tail will act as a primer from DNA synthesis by binding to the poly A tail of mRNA
• However, if the poly T tail is too long, the reverse transcriptase won’t reach
  the end of the mRNA template, and the whole DNA won’t be synthesized

Question 22

How is a gene specific primer used?

Answer 22

• A gene spefic primer is found on an open, accessible region of DNA.
• It enhances sensitivity by directing the reverse transcriptase to a specific region rather than transcribing everything
• The reverse transcriptase is therefore more likely to reach the end of the mRNA template.