8 - The Control of Gene Expression

Advantages of *in vitro* Gene Cloning

* It is extremely rapid
* It does not require living cells

Why is ‘It is extremely rapid’ an advantage of *in vitro* Gene Cloning?

A large amount of DNA can be produced in a very short time period from a very small sample.

Why is ‘It does not require living cells’ an advantage of *in vitro* Gene Cloning?

* Only a base sequence of DNA that can be amplified is needed.
* There are no complex culturing techniques needed.

What are the advantages of *in vivo* Gene Cloning?

* Useful for introducing a gene into another organism
* Almost no risk of contamination
* Very accurate
* It cuts out specific genes
* Produces transformed bacteria that can be used to produce large quantities of gene products

Why is ‘Useful for introducing a gene into another organism’ an advantage of *in vivo* Gene Cloning?

Using vectors such as plasmids allow the gene to be delivered to any organism through gene therapy.

Why is ‘Almost no risk of contamination’ an advantage of *in vivo* Gene Cloning?

Gene has been cut out by restriction endonucleases to create complementary sticky ends to the plasmid therefore the plasmid cannot take up contaminant DNA.

Why is ‘Very accurate’ an advantage of *in vivo* Gene Cloning?

The DNA copied has very few to no errors at all. Mutations arising is very rare.

Why is ‘It cuts out specific genes’ an advantage of *in vivo* Gene Cloning?

Very precise procedure as it only copies a specific gene not the whole DNA sequence.

Why is ‘Produces transformed bacteria that can be used to produce large quantities of gene products’ an advantage of *in vivo* Gene Cloning?

Commercial/medical use e.g. insulin

DNA fragment

The DNA that is being copied

Primers

Short sequences of nucleotides that have a set of bases complementary to those at one end of each of the two DNA fragments.

DNA polymerase

Enzyme that joins nucleotides together

What type of DNA polymerase is used in PCR?

Taq polymerase

Why is Taq polymerase used?

It is thermostable enzyme that can withstand the high temperatures required for PCR.

What is the optimum temperature of Taq polymerase?

Around 72°C

Thermocycler

A computer-controlled machine that varies in temperature precisely over a period of time.

Annealing

When the temperature is lowered to enable the DNA primers to attach to the template DNA.

What does PCR stand for?

Polymerase chain reaction

What are the stages of PCR?

1. **Denaturing** - Separation of DNA strand
2. **Annealing** - Annealing of the primers
3. **Synthesis** - Synthesis of DNA

What happens during the **denaturing** stage of PCR?

DNA fragments, primers and DNA polymerase are placed in vessels within a thermocycler. Temperature is increased to 95°C which breaks the hydrogen bonds splitting the DNA into two strands.

What temperature is used during the **denaturing** stage of PCR?

95°C

What happens during the **annealing** stage of PCR?

It is cooled to 55°C, causing primers to anneal to their complementary bases at the end of the DNA fragments. Primers provide the starting sequence for DNA polymerase to begin copying DNA whilst preventing the two strands from rejoining.

What temperature is used during the **annealing** stage of PCR?

55°C

What happens during the **synthesis** stage of PCR?

It is heated to 72°C. Beginning at the primer on both strands, DNA polymerase adds complementary nucleotides along each separated DNA strand until it reaches the end of the chain.

What temperature is used during the **synthesis** stage of PCR?

72°C

By what factor does the number of DNA strands increase each temperature cycle in PCR?

x2 / doubles

What are stages involved in making a protein using genetic modification?

1. **Isolate** the gene that makes the desired protein
2. **Insert** the DNA into a vector (plasmid)
3. **Transform** incorporate the gene into the desired organism using a vector (add the plasmids to bacteria)
4. **Identify** the transgenic cells
5. **Clone** the population of cells

What technique is used in *in vitro* gene cloning?

PCR

Why must the same restriction endonuclease be used on the target gene and plasmid?

To create complementary sticky ends

DNA ligase

Creates phosphodiester bonds between the bases in the complementary sticky ends of the target gene and plasmid.

What are the issues with **insertion** in *in vivo* gene cloning?

1. The plasmid sticky ends might ‘stick’ to each other, not to the other gene you are trying to insert.
2. The sticky ends of the gen you’re trying to insert might stick to each other, creating a loop that isn’t associated with the plasmid DNA.
3. RNA polymerase and transcriptional factors need a promoter region on the DNA to recognise where to start transcription.

What ‘instructions’ do RNA polymerase and transcriptional factors need to carry out transcription?

* Promoter added at the start of the gene of interest
* Terminator base sequence added at the end of the gene of interest

What are the stages of **transformation** in *in vivo* gene cloning?

* Mix plasmids and bacteria in medium containg Ca2+ ions
* Modify the temperature

Why is the temperature modified during **transformation** in *in vivo* gene cloning?

To increases membrane permeability

Approximately what percentage of bacteria will take up the plasmids in *in vivo* gene cloning?

1%

How do you know which bacteria have taken up the plasmids?

* Select a plasmid that has the first antibiotic resistance gene
* Grow the bacteria in a medium containing the anibiotic
* If the bacterium has taken up the plasmid it will have the resistance gene, so it will SURVIVE in the antibiotic solution

How do you know which plasmids have taken up the gene in *in vivo* gene cloning?

* Choose a plasmid with the restriction endonuclease site within the second antibiotic resistance gene
* Grow the bacteria in a medium containg the antibiotic
* If the original plasmid reforms the second antibiotic resistance gene will work so the bacteria survive
* Recombinant/transgenic plasmid will DIE

What is replica plating?

Replica plating is a technique used to transfer bacterial colonies from one agar plate to another. This creates a copy of the bacteria present.

Why is replica plating important?

It produces a copy of the bacteria before they are tested with the second antibiotic which will kill the plasmids that have taken up the target gene. These plasmids can then be isolated and cloned.

What plasmids are disgarded when fluorescent markers are used to test whether they have taken up the target gene?

The fluorescent ones

What plasmids are disgarded when enzyme markers are used to test whether they have taken up the target gene?

Ones that change the colour of the agar

What are marker genes?

Genes of interest are inserted into the middle of them causing inactivation.

What are examples of marker genes?

* Antibiotic resistance genes
* Fluorescent markers
* Enzyme markers

What methods are there for **isolating** target genes?

* Restriction endonucleases
* Reverse transcriptase
* Artificially synthesising genes

What are pallindromic sites?

Sites which have DNA base sequences that are read the same forwards as backwards.

What are restriction sites?

Specific pallindromic sites where restriction endonucleases cut DNA

What conditions are necessary for restriction endonucleases to be used to **isolate** a target gene?

There must be a restriction site either side of the target gene

What will the target gene be left with if restriction endonucleases are used?

Sticky ends

How can reverse transcriptase be used to **isolate** a target gene?

Transcribes mRNA into cDNA (complimentary DNA)

What does cDNA not have?

Introns

How do you artificially synthesise genes?

* Use a ‘gene machine’ to make DNA from scratch
* Join about 25 nucleotides together at once
* Forms an oligonucleotide which can be joined together to synthesis genes
* Design your own gene/protein

Oligonucleotide

An oligonucleotide is a short chain of nucleotides

Transgenic Cells

Cells whose genome has been altered by the introduction of one or more foreign DNA sequences from another species by artificial means.

Why is the genetic code degenerate?

* Multiple codons code for the same amino acids
* This happens because there are multiple tRNA molecules for each amino acid
* The amino acid can attach to any of these tRNA molecules which each have a different anticodon that can bind to different codon sequences