Amplifying Dna Fragments

Front 1

One you’ve got a fragment of DNA, you’ll probably want to make more copies of it - this is called gene cloning.

Back 1

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Front 2

What are the two techniques for gene cloning?

Back 2

• In vivo cloning

• In vitro cloning

Front 3

What are the 3 parts to In vivo cloning?

Back 3

1. Making recombinant DNA

2. Transforming cells

3. Identifying transformed cells

Front 4

Describe how to do part 1 of In vivo cloning: making recombinant DNA?

Back 4

1. The vector DNA is isolated

2. The vector DNA is cut open using the same restriction endonuclease that was used to isolate the DNA fragment - this means that the sticky ends of the vector DNA are complementary to the sticky ends of the DNA fragment

3. The vector DNA and DNA fragment are mixed together + DNA ligase is added

NB - DNA ligase joins the sticky ends of the fragment to the sticky ends of the vector DNA (process called Ligation)

4. The new DNA (vector DNA + DNA fragment) is called recombinant DNA

Front 5

Define – a vector? (in terms of gene technologies)

Back 5

Something that’s used to transfer DNA into a cell

Front 6

Give some examples of things that can be used as vectors?

Back 6

• plasmids (small, circular DNA molecules in bacteria)

• bacteriophages (viruses that infect bacteria)

Front 7

Describe how to do part 2 of in vivo cloning: Transforming cells?

Back 7

If plasmid used as vector:

• host cells are encouraged to take up the plasmid with recombinant DNA - transferring the gene into the cells

• Bacteria can be encouraged to take up DNA by electroporation - given an electric shock, creates holes in the cell through which DNA pass

If Bacteriophage used as vector:

1. bacteriophage infects the host bacterium by injecting its DNA into it

2. the phage DNA (with the target gene in it) integrates into the bacterial DNA

NB - host cells that take up the vectors containing the target gene are said to be transformed

Front 8

What do you call a host cell that takes up a vector containing a target gene?

Back 8

A transformed cell

Front 9

Describe how to do step 3 of in vivo cloning: Identifying transformed cells?

Back 9

NB - only around 5% of host cells will take up the vector DNA, so it’s important to be able to identify which cells have been transformed - Marker genes are used

1. A marker gene can be inserted into vectors at the same time as the target gene - any transformed host cells will contain this marker gene

2. Host cells grown on agar plates, each cell divides and replicates its DNA, creating a colony of cloned cells

3. Either:

• Agar can contain an antibiotic, the marker gene can code for antibiotic resistance, only transformed cells with the marker gene will survive and grow

• Marker gene can code for fluorescence, when the agar plate is placed under UV light, only transformed cells will fluoresce

4. Identified transformed cells are allowed to grow more - producing lots of copies of the cloned gene

Front 10

Describe how to do step 3 of in vivo cloning: Identifying transformed cells?

Back 10

NB - only around 5% of host cells will take up the vector DNA, so it’s important to be able to identify which cells have been transformed - Marker genes are used

1. A marker gene can be inserted into vectors at the same time as the target gene - any transformed host cells will contain this marker gene

2. Host cells grown on agar plates, each cell divides and replicates its DNA, creating a colony of cloned cells

3. Either:

• Agar can contain an antibiotic, the marker gene can code for antibiotic resistance, only transformed cells with the marker gene will survive and grow

• Marker gene can code for fluorescence, when the agar plate is placed under UV light, only transformed cells will fluoresce

4. Identified transformed cells are allowed to grow more - producing lots of copies of the cloned gene

Front 11

There’s a final important step in in vivo cloning, if you want the transformed host cells to produce the protein coded for by the target gene.

What is it?

Back 11

Make sure that the vector contains specific promoter and terminator regions

Promoter regions - DNA sequences that tell the enzyme RNA polymerase where to start producing the mRNA

Terminator regions - DNA sequences that tell the enzyme RNA polymerase where to stop producing mRNA

Front 12

There’s a final important step in in vivo cloning, if you want the transformed host cells to produce the protein coded for by the target gene.

What is it?

Back 12

Make sure that the vector contains specific promoter and terminator regions

Promoter regions - DNA sequences that tell the enzyme RNA polymerase where to start producing the mRNA

Terminator regions - DNA sequences that tell the enzyme RNA polymerase where to stop producing mRNA

Front 13

There’s a final important step in in vivo cloning, if you want the transformed host cells to produce the protein coded for by the target gene.

What is it?

Back 13

Make sure that the vector contains specific promoter and terminator regions

• Promoter regions - DNA sequences that tell the enzyme RNA polymerase where to start producing the mRNA

• Terminator regions - DNA sequences that tell the enzyme RNA polymerase where to stop producing mRNA

Without the right promoter regions, the gene won’t be transcribed by the host cell and a protein won’t be made

These regions may already be present in the vector DNA or may have to be added in

Front 14

How do you do in vitro cloning via the polymerase chain reaction (PCR) method?

Back 14

1. Reaction mixture set up, contains: the DNA sample, free nucleotides, primers, DNA polymerase

2. The DNA mixture is heated to 95° C -> breaks H-bonds between the two DNA strands

3. The mixture is cooled to 55°C so that the primers can bind (anneal) to the strands

4. Reaction mixture is heated to 72°C so that DNA polymerase can work - DNA polymerase lines up free DNA nucleotides alongside each template strand, CBP forms complimentary strands

5. 2 new copies of the DNA fragment are formed and one cycle of PCR is complete

6. The cycle starts again - the mixture is heated to 95°C, this time all four strands are used as templates, forming 4 fragments

(See hint for picture)

Hint 14

Front 15

What are - Primers?

Back 15

Short pieces of DNA that are complimentary to the bases at the start of the fragment you want to copy

Front 16

In in vitro cloning, ‘the reaction mixture is heated to 72°C so that DNA polymerase can work.’

How is it not denatured?

Back 16

Use Taq polymerase - comes from bacteria in hot springs, able to withstand high temperatures

Front 17

Define- in vitro cloning?

Back 17

Gene copies are made outside of a living organism

Front 18

Define - in vivo cloning?

Back 18

Gene copies are made within a living organism

-> as the organism grows and divides, it replicates the DNA, creating multiple copies of the gene

Front 19

Picture to help visualise the repeats of multiple PCR cycles

Back 19

Back (Definition)