Recombinant DNA technology (A-level only)
AQA
A-Level
Recombinant DNA is DNA made by joining together DNA from two different organisms, usually from different species. The sequence of operations is fixed and worth learning as a whole. The required gene is isolated - cut out with restriction endonucleases, made from mRNA using reverse transcriptase, or built in a gene machine. Promoter and terminator regions are added so that the host's RNA polymerase knows where to start and stop. The fragment is inserted into a vector, normally a plasmid or a virus, using the same restriction enzyme to create complementary sticky ends and DNA ligase to seal the sugar-phosphate backbones. The vector carries the DNA into a host cell; a cell that takes it up is transformed, and a whole organism containing DNA from another species is transgenic.
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Objectives
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Exam Tips
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Pitfalls
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Key Terms
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Mark Points
Subtopics in this area
Recombinant DNA technology involves the transfer of fragments of DNA from one organism, or species, to another.
Since the genetic code is universal, as are transcription and translation mechanisms, the transferred DNA can be translated within cells of the recipient (transgenic) organism.
Fragments of DNA can be produced by several methods, including: conversion of mRNA to complementary DNA (cDNA), using reverse transcriptase using restriction enzymes to cut a fragment containing the desired gene from DNA creating the gene in a ‘gene machine’.
Fragments of DNA can be amplified by in vitro and in vivo techniques.
The principles of the polymerase chain reaction (PCR) as an in vitro method to amplify DNA fragments.
The culture of transformed host cells as an in vivo method to amplify DNA fragments.
The addition of promoter and terminator regions to the fragments of DNA.
The use of restriction endonucleases and ligases to insert fragments of DNA into vectors.
Transformation of host cells using these vectors.
The use of marker genes to detect genetically modified (GM) cells or organisms.
Recombinant DNA technology (A-level only) Revision Guide
Learning Objectives
What you need to know and understand
- Outline the full sequence by which a gene from one species ends up being expressed in another.
- Distinguish between a vector, a host cell and a transgenic organism, using each term correctly.
- Explain why promoter and terminator regions are added to the fragment before it is placed in a vector.
- State two distinct reasons, code and mechanism, why a bacterium can express a human gene.
- Explain why cDNA rather than chromosomal DNA is transferred when a eukaryotic gene is to be expressed in a bacterium.
- Suggest, naming a specific organelle or mechanism that bacteria lack, why some human proteins cannot be produced in bacteria.
- Describe how reverse transcriptase is used to produce a DNA fragment from mRNA, naming the substrates needed.
- Explain why a gene made as cDNA or in a gene machine can be expressed by a bacterium when a chromosomal copy cannot.
- Describe how a restriction endonuclease produces sticky ends, naming the site it recognises and the bond it hydrolyses.
- Distinguish in vitro from in vivo amplification and name the method used in each case.
- Explain why culturing transformed host cells increases the number of copies of an inserted fragment.
- Compare PCR and in vivo amplification on speed, quantity, accuracy and whether protein is produced.
- Describe the three temperature stages of one PCR cycle and state what happens to the DNA at each.
- Explain why a thermostable polymerase and short primers are both essential to the reaction.
- Calculate the number of DNA molecules present after a stated number of cycles, starting from a given number.
- Describe how transformed cells are identified using marker genes and insertional inactivation.
- Explain why the number of copies of a plasmid increases as a bacterial population grows.
- Compare in vivo amplification with PCR in terms of quantity of product, accuracy and whether protein is obtained.
- Name the regions added to either end of a DNA fragment and state what each does during transcription.
- Explain why a gene transferred without a promoter produces no protein in the host cell.
- Suggest how choosing a particular promoter controls which tissue of a transgenic organism expresses the gene.
- Explain why the same restriction enzyme must be used on both the fragment and the vector.
- Describe the two kinds of bond involved in inserting a fragment into a plasmid and state which enzyme, if any, forms each.
- Explain why using a single restriction enzyme allows a fragment to be inserted in either orientation.
- Describe how bacterial cells are treated so they will take up a recombinant plasmid, and explain why the treatment works.
- Name a suitable vector and transformation method for a plant cell and for an animal cell.
- Explain why a selection step is always required after transformation.
- Explain the purpose of a marker gene in terms of which cells can be identified after transformation.
- Describe how an antibiotic resistance marker is used to separate transformed from untransformed cells.
- Suggest why fluorescent or enzyme markers are now preferred to antibiotic resistance genes.
Marking Points
Key points examiners look for in your answers
- one mark for recombinant DNA being DNA from two different organisms or species joined together
- one mark for the fragment being isolated by a named method before transfer
- one mark for the fragment being inserted into a vector such as a plasmid or a virus
- one mark for the vector carrying the DNA into the host cell, which is then described as transformed or transgenic
- one mark for a named useful product, such as a human protein produced by bacteria or by a genetically modified animal
- The genetic code is universal, meaning the same triplets or codons code for the same amino acids in almost all species.
- The mechanism of transcription is fundamentally the same in both organisms.
- The mechanism of translation is the same, as bacteria possess ribosomes to translate the mRNA.
- Bacteria cannot splice pre-mRNA because they lack spliceosomes, so introns are not removed.
- Bacteria lack a Golgi apparatus, so the protein cannot undergo post-translational modifications like glycosylation.
- Bacteria lack eukaryotic promoter recognition, which can prevent the transcription of unmodified eukaryotic genes.
- one mark for using reverse transcriptase with free DNA nucleotides to make cDNA from mRNA
- one mark for explaining that cDNA contains no introns because the mRNA has already been spliced
- one mark for restriction endonucleases cutting the DNA at specific base sequences, that is at recognition or restriction sites, accepting palindromic sequences
- one mark for a staggered cut producing sticky ends, short exposed single-stranded sections of bases
- one mark for the gene machine building the gene from a known base sequence, free of introns
- one mark for using PCR to amplify the DNA or sample, or a correct description of amplification
- one mark for identifying PCR as in vitro, taking place outside living cells
- one mark for the in vivo route being insertion of the fragment into a vector and culture of transformed host cells
- one mark for the plasmid being copied each time the host cell divides, so copy number rises with the population
- one mark for a valid comparison, such as PCR being faster and needing a smaller sample, while in vivo culture also yields the protein and larger quantities
- one mark for heating to about 95 degrees Celsius to break the hydrogen bonds and separate the two DNA strands
- one mark for cooling so that primers with complementary base sequences bind to the ends of the target strands
- one mark for DNA polymerase joining free DNA nucleotides to form complementary strands, at about 72 degrees Celsius
- one mark for the number of DNA molecules doubling each cycle, so n cycles produce two to the power n copies
- one mark for the use of the product, such as providing enough DNA from a small sample to sequence, screen or fingerprint
- one mark for the vector being taken up by host cells, which are then described as transformed
- one mark for a primary marker gene identifying cells that have successfully taken up a plasmid
- one mark for a secondary marker gene identifying recombinant plasmids via insertional inactivation
- one mark for culturing only the transformed cells under suitable conditions of nutrients, temperature, pH and oxygen
- one mark for the plasmid being copied each time the cell divides by binary fission, increasing fragment copies
- one mark for the protein also being produced, because promoter and terminator regions were added to the fragment
- one mark for naming the region added at the start of the gene as the promoter and the region at the end as the terminator
- one mark for the promoter being the binding site for RNA polymerase, or for transcription factors, so that transcription is initiated
- one mark for the terminator causing RNA polymerase to stop and detach, releasing the mRNA
- one mark for explaining that without a promoter the gene is not transcribed, so the protein is not produced
- one mark for explaining that without a terminator transcription may continue past the gene, producing run-on RNA rather than correctly ended mRNA
- one mark for a promoter determining in which cells or tissue the gene is expressed
- one mark for using a restriction endonuclease to cut the DNA at a specific palindromic recognition sequence
- one mark for using the same restriction enzyme on the vector, so that complementary sticky ends are produced
- one mark for complementary bases on the sticky ends pairing, with hydrogen bonds forming between them
- one mark for DNA ligase joining the sugar-phosphate backbones by catalysing the formation of phosphodiester bonds
- one mark for describing the product as recombinant DNA or a recombinant plasmid
- one mark for mixing host cells and recombinant plasmids in a calcium ion solution and applying heat shock
- one mark for explaining that this makes the cell wall or cell-surface membrane more permeable, so the plasmid can enter
- one mark for stating that only a small proportion of the host cells are transformed
- one mark for naming an alternative vector or method, such as a virus, a liposome, electroporation or the Agrobacterium plasmid
- one mark for the need to identify transformed cells afterwards, using a marker gene
- One mark for the purpose of the marker gene: to show that the plasmid has been taken up, or to identify transformed cells.
- One mark for explaining that the marker gene is inserted into the vector, allowing detection of cells that have taken up the plasmid.
- One mark for naming a marker and its detection method, such as fluorescence under ultraviolet light or a colour change with an enzyme marker.
- One mark for explaining how insertional inactivation (e.g., disrupting a second marker gene) distinguishes recombinant plasmids from empty plasmids.
Examiner Tips
Expert advice for maximising your marks
- 💡Learn the sequence as five steps - isolate, add promoter and terminator, insert into vector, transform, culture - and you can answer almost any version of this question.
- 💡Use 'transformed' for cells and 'transgenic' for whole organisms; the terms are marked separately.
- 💡Name the product and the host when you can; a specific example often carries the final mark.
- 💡Two distinct marks are often available for stating the code is universal and the mechanisms are universal; write both.
- 💡When asked why a bacterium might fail to make a functional human protein, name a missing structure (e.g., Golgi apparatus) and its function (e.g., glycosylation).
- 💡If a question gives you a choice of method, cDNA is the safe answer for a eukaryotic gene because of the introns.
- 💡Use the phrase 'specific base sequence' or 'recognition site' whenever restriction enzymes appear - it is where the mark sits.
- 💡Say which bond is broken or formed; the examiner is checking you know phosphodiester bonds from hydrogen bonds.
- 💡Fix the terms by their literal meaning: in vitro is in glass, in vivo is in the living.
- 💡Comparison questions want a named criterion - speed, quantity, accuracy or whether protein is produced.
- 💡If the question says 'amplify', the first mark is almost always the word PCR or a description of doubling.
- 💡Give a temperature with a reason attached; a bare list of numbers rarely scores.
- 💡For calculations use two to the power n, and check whether the question counts the starting molecules in the total.
- 💡Mention that the primers set the boundaries of the region copied - it explains why PCR is specific.
- 💡Mention the selection step; an answer that jumps straight from transformation to culture misses an easy mark.
- 💡Say binary fission, not mitosis, whenever bacterial division is involved.
- 💡Distinguish between primary markers for plasmid uptake and secondary markers for identifying recombinant plasmids.
- 💡On a diagram of a DNA fragment, the region before the gene is usually the promoter and the region after it the terminator, but check the labels rather than assuming.
- 💡Tie each region to RNA polymerase - starting for the promoter, detaching for the terminator.
- 💡If asked why a transgenic gene is not expressed, a missing promoter is usually the intended answer.
- 💡Two enzymes, two different bonds: restriction endonuclease breaks phosphodiester bonds, ligase forms them. Say which each time.
- 💡The word 'same' before 'restriction enzyme' is crucial; without it the complementary sticky ends have no explanation.
- 💡Remember that using a single restriction enzyme to cut both ends allows the fragment to insert in either orientation.
- 💡Give both halves of the bacterial method - calcium ions and heat shock - and say what they do to the membrane.
- 💡Always add the sentence that only a small proportion of cells are transformed; it sets up the marker gene question that usually follows.
- 💡Match the method to the organism: plasmids and heat shock for bacteria, viruses or liposomes for animal cells.
- 💡When asked how transformed cells are identified, state both the marker used and the specific method of detection (e.g., UV light for GFP).
- 💡If asked for a modern alternative to antibiotic resistance, suggest fluorescent or enzyme markers and state that they prevent the spread of antibiotic resistance to pathogenic bacteria.
Common Mistakes
Pitfalls to avoid in your exam answers
- calling the recipient organism 'genetically mutated' instead of transformed or transgenic
- describing the gene as injected straight into a bacterium with no vector at all
- confusing the vector with the host cell, so calling the bacterium the vector
- writing that the bacteria 'make the gene' rather than the protein for which the gene codes
- leaving out promoter and terminator regions, so nothing explains how the gene is expressed
- Writing 'DNA is universal' on its own, which is not accepted unless qualified as the genetic code.
- Stating 'the genetic code is degenerate' as the reason a bacterium can use a human gene; this is incorrect and rejected.
- Claiming bacteria lack transcription factors entirely, rather than lacking specific eukaryotic promoter recognition.
- Forgetting introns and claiming a bacterium can express any human gene taken straight from a chromosome.
- saying reverse transcriptase makes mRNA from DNA, which is the wrong direction
- saying restriction enzymes cut at random, or that they simply 'cut the gene out', with no recognition sequence named
- describing sticky ends as complementary amino acids or as complementary genes
- choosing any cell to extract mRNA from, when the mark is for a cell that actively produces the protein
- saying the restriction enzyme breaks the hydrogen bonds between the strands, when it hydrolyses phosphodiester bonds in the backbone
- using in vitro and in vivo the wrong way round
- describing PCR as taking place inside a bacterium
- saying PCR produces protein, when it produces only DNA
- claiming PCR 'makes a gene', when it copies a fragment that must already be present
- giving a comparison with no criterion, such as 'PCR is better', with nothing measured
- saying DNA helicase separates the strands, when in PCR heat alone breaks the hydrogen bonds
- using ordinary human DNA polymerase, which would be denatured at 95 degrees Celsius
- saying the primers are RNA, or that they are complementary to the whole gene rather than to its ends
- calculating copies as n multiplied by two rather than two to the power n, or forgetting the original molecule
- writing that PCR amplifies protein or mRNA
- assuming every host cell takes up the vector; correction: describe the selection step using marker genes
- saying the bacteria copy the plasmid by mitosis; correction: state that prokaryotes divide by binary fission
- stating a single marker gene confirms the target DNA is present; correction: a single marker only confirms plasmid uptake, a second marker (insertional inactivation) is needed to confirm the insert
- writing 'grown in a fermenter' with no conditions named; correction: specify conditions like suitable temperature, pH and nutrients
- saying the promoter is where translation begins, or confusing it with the start codon on the mRNA (correct: the promoter is a DNA sequence where transcription is initiated)
- stating that RNA polymerase binds to the terminator region (correct: RNA polymerase binds the promoter and is released at the terminator)
- believing the promoter and terminator are never transcribed (correct: the terminator may be transcribed into RNA, though it is not translated into protein)
- writing that adding a promoter 'makes more protein' with no mention of transcription (correct: link the promoter to initiation of transcription)
- adding only a promoter and assuming transcription will stop by itself (correct: a terminator is also needed to release RNA polymerase and end transcription)
- using different restriction enzymes on the gene and the plasmid and still expecting the ends to anneal; correction: specify the same enzyme must be used
- saying DNA ligase forms hydrogen bonds; correction: ligase catalyses phosphodiester bonds, while hydrogen bonds form spontaneously between complementary bases
- saying the restriction enzyme cuts in the middle of the gene; correction: state that enzymes cut at chosen recognition sites which must flank the target gene to avoid destroying it
- stopping at base pairing and never mentioning ligase; correction: always include ligase to seal the sugar-phosphate backbone
- saying the plasmid enters by active transport, when it crosses a membrane made temporarily permeable
- confusing transformation with transcription or translation, which are stages of protein synthesis
- assuming every host cell is transformed, so no selection is needed
- calling the plasmid a host cell, or calling the bacterium the vector
- describing heat shock without the calcium ion step, so nothing explains the increased permeability
- Saying the marker gene 'marks the gene', without stating that it identifies the cells that have taken up the plasmid.
- Confusing cells with empty plasmids and recombinant plasmids; remember that a single marker only shows plasmid uptake, while insertional inactivation of a second marker confirms the desired gene is present.
- Naming the enzyme marker as lactase instead of β-galactosidase, or forgetting that fluorescent markers require ultraviolet light for detection.