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Recombinant DNA technologyAQA A-Level Biology: Revision notes

Section 1

Transferring genes

Recombinant DNA technology transfers fragments of DNA from one organism or species to another. The recipient is a transgenic organism. It works because the genetic code is universal, as are transcription and translation mechanisms, so the transferred DNA can be translated in the recipient cells.

Key termsrecombinant DNAtransgenic

Section 2

Producing the DNA fragment

A gene can be obtained in three ways:

  • cDNA: reverse transcriptase makes DNA from mRNA. The cDNA has no introns, and cells that make a lot of the protein have a lot of the mRNA.
  • Restriction endonucleases cut the gene from DNA at specific recognition sequences, often leaving sticky ends.
  • A gene machine builds the gene from the base sequence, without needing cells.
Key termscDNAreverse transcriptaserestriction endonuclease

Section 3

In vitro amplification: PCR

The polymerase chain reaction copies DNA in vitro. Each cycle has three steps:

  1. Heat to about 95 °C: hydrogen bonds break and strands separate.
  2. Cool to about 55 °C: primers anneal to the ends of the target sequence.
  3. Heat to about 72 °C: heat-stable DNA polymerase joins nucleotides to the primers, making new complementary strands.

The number of copies doubles each cycle: after nn cycles there are 2n2^n copies. After 10 cycles from one molecule: 2¹⁰ = 1024.

Key termsPCRprimerDNA polymerase
Exam tip

Always link the temperature to what is happening: strands separate, primers anneal, new strands are made.

Section 4

In vivo amplification: transformed host cells

The gene fragment is given a promoter region, so RNA polymerase binds, and a terminator region to stop transcription. It is cut with a restriction endonuclease and joined into a vector, for example a plasmid, cut with the same enzyme, using DNA ligase. The vector is used to transform host cells, which are then cultured, copying the gene each time they divide.

Key termsvectorligasepromoterterminator

Section 5

Marker genes

Not all cells take up the vector. Marker genes on the vector, such as antibiotic resistance or a gene for fluorescence, are used to detect GM cells or organisms. Specific marker genes need not be recalled. If the gene is inserted inside a marker gene, that marker is disrupted, so cells with the gene are identified by the loss of that marker.

Key termsmarker geneGM
Common mistake

Do not say that antibiotic resistance makes bacteria take up the plasmid. It only identifies the cells that did.

That's the notes covered.

Carry on to the next subtopic.

Exam questions on Recombinant DNA technology

  1. Beta cells of the human pancreas make large amounts of mRNA for insulin. Researchers want bacteria to make human insulin. Bacterial cells cannot remove introns from RNA. The researchers use mRNA from the beta cells as the starting material to obtain a copy of the insulin gene.
    Explain why the human insulin gene can be transcribed and translated to make insulin in the bacterial cell.2 marks
  2. A plasmid vector has a single recognition site for the restriction endonuclease EcoRI, which cuts DNA leaving short single-stranded ends. A fragment of human DNA containing a gene is cut out of a chromosome using the same enzyme and then mixed with the cut plasmids.
    Explain why EcoRI cuts DNA only at one specific base sequence.2 marks
  3. A forensic laboratory has only a few molecules of DNA from a crime scene. It uses the polymerase chain reaction (PCR) to make many copies of a short target sequence before analysis. The reaction mixture contains the DNA, primers, free nucleotides and a heat-stable DNA polymerase.
    Describe what happens in one cycle of PCR.3 marks
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Written by the Exaim team, led by Shaun Daswani (Head of Upper Secondary, Improve ME Institute; MSc Financial Mathematics, Imperial College London; BSc, UCL) and Jason Daswani (operational lead, Improve ME Institute; LSE).