Enzyme and restriction mapping

Question 1

What is a recombinant protein?

Answer 1

Protein produced from the transcription and translation of recombinant DNA

Question 2

What is recombinant DNA?

Answer 2

DNA produced via bringing together DNA from multiple sources to create sequences not found in the genome

Question 3

Name some examples of recombinant proteins

Answer 3

• Insulin
• Interferon - Group of signaling proteins made and released by host cells in response to the presence of several viruses
• G-CSF (Granulocyte-colony stimulating factor) - Glycoprotein that stimulates bone marrow to produce and then release granulocytes, a type of white blood cell, into the bloodstream.

Question 4

What is a transgenic organism?

Answer 4

An organism with an altered genome

Question 5

What are some of the uses of transgenic organisms?

Answer 5

• Produce disease models
• Improve agricultural yields - genetically modify crops to be resistant to certain pests

Question 6

What is the function of a nuclease?

Answer 6

An enzyme that cleaves nucleic acids by the phosphodiester bonds between the nucleotides

Question 7

What are the two different types of nuclease enzyme?

Answer 7

• Ribonuclease (RNase): Cleaves RNA
• Deoxyribonuclease (DNase): Cleaves DNA

Question 8

What are the 2 types of deoxyribonuclease?

Answer 8

• Exonuclease: Cleaves the DNA from the end of the molecule
• Endonuclease: Cleaves the DNA from within the nucleotide chain

Question 9

Why are restriction endonucleases (restriction enzymes) called that?

Answer 9

Because they are expressed in bacteria and their function in bacteria is to limit (restrict) the transfer of nucleic acids from the bacteria to phages.

Question 10

Explain the basic mechanism for how a restriction endonuclease works?

Answer 10

• They recognise a specific sequence
• Then cleave that specific sequence at a recognition site

Question 11

Name an example of a restriction enzyme. What is the specific recognition sequence that this restriction enzyme recognises?

Answer 11

• EcoRI
• Specific recognition sequence: G/AATTC
• Palindromic complementary sequence: CTTAA/G

Question 12

What does it mean when it is said that recognition sequences are “palindromic?”

Answer 12

• It means that they can be read the same way in both directions - from 5’ to 3’ or from 3’ to 5’
• It also means that every recogntion sequence also has a complementary palindromic sequence which is the same sequence as the recognition sequence but is read in the opposite direction.

Question 13

Where specifically does EcoRI cleave within the recognition sequence?

Answer 13

It cleaves between the Guanine and adenine nucleotides of the recogniton sequence

Question 14

What are the effects of the cleavage from EcoRI on the recognition sequence?

Answer 14

• It creates a 5’ overhang on both the recognition sequence and the palindromic sequence
• Due to the hydrolysis rection that occured on the 3’ end of the guanine nucleotides there are OH groups attached while at the 5’ end of the adenine nucleotides there are phosphate groups attached

Question 15

What is an overhang?

Answer 15

• A stretch of unpaired nucleotides within a DNA molecule
• Can be 3’ or 5’ overhang

Question 16

Apart from overhang what other effect can a restriction endonuclease produce within the recognition sequence?

Answer 16

• Can also produce blunt ends which is when both strands of a DNA molecule end in a base pair.

Question 17

What is a restriction map?

Answer 17

A map of known restriction sites within a sequence of DNA.

Question 18

Describe a method that can be used to produce a restriction map for a particular plasmid?

Answer 18

• You have a plasmid of known size within a set of test tubes
• To each of the plasmid test tubes you add a different combinations of restriction endonuclease - E.g. In one test tube you add EcoRI; in another you add BamHI and in the final test tube you add both restriction endonucleases.
• You then run PCR for each of the different test tubes at 37 °C for a period of time.
• After this you then use gel electrophoresis to separate the fragments produced within each test tube onto a DNA gel.
• You then work out the size of each of the fragments produced from each restriction enzyme and use these to produce a restriction map

Question 19

What does the result of the EcoRI lane for gel electrophoresis tell you about how it digests the pDTL3 plasmid?

Answer 19

• EcoRI only has one band at 13kb which is the same size as the plasmid
• This means that it didn’t digest the particular sample or if it did it only cleaved the sample at one position

Question 20

What does the result for the BamHI lane for gel electrophoresis tell you about how it digets the pDTL3 plasmid?

Answer 20

• BamHI produced 2 fragments, one of which is 6kb and the other 7kb
• This means that it cleaved the pDTL3 plasmid twice

Question 21

What are size standards and why are they run on the DNA gel during gel electrophoresis?

Answer 21

• Size standards are DNA fragments which we know the length of.
• They are used to determine the length of the different DNA fragments produced from the restriction enzyme-plasmid reactions so a restriction map can then be produced

Question 22

How can the results from the BamHI lane for gel electrophoresis be used to locate the restriction sites for BamHI on a pDTL3 restriction map?

Answer 22

• You know that BamHI produced a 6kb fragment and a 7kb fragment
• You also know that the entire pDTL3 is 13kb in size
• With this information on a restriction map you simply place one of the restriction sites on the top of the map.
• You can then work out where on the map the other restriction site is because you know the length of the two fragments produced.

Question 23

Can the results from the EcoRI lane be used to locate where its restriction site/s are on the pDTL3 restriction map?

Answer 23

No because the EcoRI only produced one fragment which was the same length as the plasmid so you have no indication of where exactly EcoRI cleaved the plasmid

Question 24

In this example what can be used to locate the restriction site for EcoRI on the pDTL3 restriction map? Explain how it can be used to do this

Answer 24

• You have to look at the size of the fragments produced when both restriction endonucleases were used.
• In this example the 6Kb fragment is still produced meaning that within that fragment there is no EcoRI restriction site present.
• However, the 7Kb band produced by BamHI is now split into a 3 and a 4Kb fragment which means the EcoRI restriction site is located within the 7kb fragment produced by BamHI

Question 25

Restriction maps can also be produced for linear DNA. What is the difference between the way fragments from gel electrophoresis are used to determine how a particular restriction enzyme digests linear DNA compared with a plamsid?

Answer 25

* For linear DNA if a restriction endonuclease produces 2 fragments it only cleaves the DNA molecule at one position rather than 2 like it would indicate if it were a plasmid

Question 26

How does the sickle cell anaemia mutation affect the activity of the restriction enzyme Dde1?

Answer 26

* The normal 𝛃-globin gene (𝛃^A) contains the recognition sequence for Dde1: CTGAG * However, the sickle cell anaemia mutation causes this sequnce to change to CTGTG in the mutated 𝛃-globin gene (𝛃^S) which Dde1 is unable to recognise * This means that in the mutated 𝛃-globin gene Dde1 isn't able to cleave the gene at that position

Question 27

How can the change in activity in Dde1 in response to the sickle cell anaemia mutation be used to determine the chances of the offspring of two parents will be a carrier, affected or not affected by the mutation?

Answer 27

* You would take a sample of genomic DNA from blood cells of the mother and father * Then use PCR to elongate the specific DNA segment within the 𝛃-globin gene where the single nucleotide variant occurs. * You would then add Dde1 to the elongated PCR DNA sample and run the resulting DNA fragments onto DNA gel using gel electrophoresis * Then you would use the resulting fragmentation pattern on the gel to work out whether each parent is homozygous for the mutated 𝛃-globin gene; heterozygous or homozygous for the normal 𝛃-globin gene. * This can then be used to calculate the probabilty of the offspring being a carrier, affected or unaffected

Question 28

What size fragments would you expect to see on the gel electrophoresis gel if a person was homozygous for the normal 𝛃-globin gene? why is this?

Answer 28

* You would expect to see 3 fragments: * One 175 bp fragment * One 201 bp fragment * One 67 bp fragment * This is because on both copies of the gene Dde1 is able to recognise and cleave the recognition site 175 bps away from the start of the sequence * Dde1 also cleaves second recognition site 67bp away from the end of the sequence which also creates a 201bp fragment

Question 29

What size fragments would you expect to see on the gel electrophoresis gel if a person was heterozygous for the 𝛃-globin gene? why is this?

Answer 29

* You would see 4 fragments: * One 376 bp fragment * One 201 bp fragment * One 175 bp fragment * One 67 bp fragment * This is because on one of the copies of the 𝛃-globin gene Dde1 isn't able to recognise the first recognition sequence and so doesn't produce a cut at 175 bp so instead you have a 376 bp fragment * On the other copy of the gene the recognistion site is able to be recognised by Dde1 so it does cleave at 175 bp producing a 175bp fragment and a 201 bp fragment * Dde1 able to recognise second recognition sequence on both copies of the gene so 67 bp fragment produced.

Question 30

What size fragments would you expect to see on the gel electrophoresis gel if a person was homozygous for the mutated 𝛃-globin gene? why is this?

Answer 30

* You would see 2 fragments: * One 376 bp fragment * One 67 bp fragement * This is because on both copies of the gene Dde1 is unable to recognise the first recognition sequence so instead of a 175 bp fragment being produced you just have a 376 bp fragment * On both copies of the gene the Dde1 able to recognise the second recognition site so a 67 bp fragment is produced.

Question 31

Why is the Dde1 able to produce a 67 bp fragment regardless of whether it is cleaving the DNA sequence of a normal 𝛃-globin gene or a mutated one?

Answer 31

* Because there was a second Dde1 restriction site within the elongated DNA fragments * This restriction site is used as a control group to see if the Dde1 is working. * If you don’t get a DNA fragment at 67bp for every DNA segment on the gel it means the Dde1 enzyme isn’t working properly

Question 32

How do restriction enzymes help in the formation of recombinant DNA?

Answer 32

They allow specific DNA sequnces from different sources to be "cut out"

Question 33

What is DNA ligase?

Answer 33

An enzyme that forms phosphodiester bonds in the sugar-phosphate backbone at areas within a DNA strand where there is an overhang or a sticky end

Question 34

What is DNA polymerase?

Answer 34

An enzyme that can synthesise a new DNA strand using a template DNA strand to turn a single stranded DNA molecule into a double stranded DNA molecule

Question 35

What are some of the uses of DNA polymerase in the lab?

Answer 35

* Used in PCR * Used to generate DNA probes * Used to turn DNA overhangs into blunt-ends on a DNA sample so they can then be ligated together.

Question 36

What is a phosphatase enzyme?

Answer 36

Enzymes that remove phosphate groups from its substrate

Question 37

What are some of the uses of phosphatase in the lab?

Answer 37

* Used to prevent cut plasmids from resealing in a ligation reaction. * Without the phosphate group there is nothing for the 3’ OH group to reform a phosphodiester bond with meaning the plasmid can’t be joined back together.

Question 38

What is a polynucleotide kinase?

Answer 38

An enzyme that is able to phosphorylate (add a phosphate group to) a substrate using ATP as a source of energy

Question 39

What are some of the uses of polynucleotide kinases in the lab?

Answer 39

* To phosphorylate chemically synthesized DNA - so that it can be ligated to another fragment. * To sensitively label DNA so that it can be traced using: * radioactively labeled ATP * fluorescently labeled ATP

Question 40

What is reverse transcriptase?

Answer 40

* RNA dependent DNA polymerase * Synthesizes a DNA molecule complementary to a mRNA template using dNTPs

Question 41

What are some uses of reverse transcriptase in the lab?

Answer 41

* Used in quantitative PCR to find out how much RNA is present within a certain gene for example.

Question 42

Why is reverse transcriptase needed in quantative PCR?

Answer 42

Needed in qPCR as the RNA sample has to first be reverted back to DNA as there are currently no known polymerases that are able to elongate RNA strands.