Creating DNA Fragments Revision Notes | A Level AQA Biology

1. Why Creating DNA Fragments Matters

Imagine you want to produce human insulin on an industrial scale. You cannot extract enough from human tissue, and the gene for insulin is buried inside a chromosome alongside thousands of other genes. The challenge is isolating or recreating that gene precisely — and that is exactly what the three methods in this topic solve.

Producing a DNA fragment containing a desired gene is the essential first step in genetic engineering. Without a reliable fragment, you cannot insert the gene into a vector, express it in a host organism, or study its function. The three methods — using reverse transcriptase to make cDNA, cutting with restriction enzymes, and synthesising artificially in a gene machine — each suit different situations, and examiners expect you to know when and why each is used.

2. Method 1 — Converting mRNA to cDNA Using Reverse Transcriptase

The first method exploits a clever biological trick. Certain cells produce large quantities of a specific protein — for example, pancreatic beta cells produce insulin. Because they are actively transcribing the insulin gene, they contain abundant mRNA molecules with the exact sequence you need.

The enzyme reverse transcriptase — originally found in retroviruses — catalyses the synthesis of a DNA strand using an mRNA molecule as the template. This is the reverse of the normal transcription direction, hence the name. The product is a single-stranded DNA molecule called complementary DNA (cDNA), because its base sequence is complementary to the mRNA template.

Once the single-stranded cDNA is produced, DNA polymerase is used to synthesise the complementary strand, converting the single-stranded cDNA into double-stranded DNA. This double-stranded cDNA can then be inserted into a vector for expression in a host cell.

Worked example: Producing cDNA from mRNA — Step by Step

Describe the sequence of events that converts an mRNA molecule from a pancreatic beta cell into a double-stranded cDNA molecule suitable for genetic engineering.

1. Extract mRNA from cells that actively express the desired gene (e.g. insulin mRNA from beta cells). — These cells contain abundant copies of the specific mRNA because the gene is being actively transcribed. This gives you a pure, concentrated source of the correct sequence.
2. Reverse transcriptase reads the mRNA template (3' to 5') and assembles a complementary DNA strand (5' to 3') using free DNA nucleotides. — Complementary base pairing rules apply: adenine (A) in mRNA pairs with thymine (T) in DNA; uracil (U) in mRNA pairs with adenine (A) in DNA; cytosine (C) pairs with guanine (G).
3. The result is an mRNA–cDNA hybrid duplex. The mRNA strand is then degraded (often by RNase H), leaving single-stranded cDNA. — The cDNA sequence mirrors the coding sequence of the original gene, but it is now in DNA form and lacks introns — a critical advantage for expression in prokaryotic hosts.
4. DNA polymerase uses the single-stranded cDNA as a template to synthesise the complementary strand, producing double-stranded cDNA. — Double-stranded cDNA is stable and can be ligated into a vector. It is now a functional gene copy ready for cloning.

Answer: Double-stranded cDNA containing the coding sequence of the desired gene, with no introns, ready for insertion into a vector.

The critical advantage of the cDNA method is that the product contains no introns. Eukaryotic genes contain non-coding intron sequences that are spliced out of pre-mRNA before translation. Because prokaryotic host cells (such as bacteria) lack the splicing machinery to remove introns, inserting a genomic copy of a eukaryotic gene into a bacterium would produce a non-functional protein. cDNA bypasses this problem entirely, because it is made from the already-processed mRNA.

3. Method 2 — Using Restriction Enzymes to Cut a DNA Fragment

The second method works directly on genomic DNA. If you already know where a gene sits on a chromosome, you can use restriction enzymes (also called restriction endonucleases) to cut the DNA at precise locations flanking the gene, releasing the fragment you need.

Each restriction enzyme recognises a specific, short palindromic base sequence — typically 4 to 8 base pairs long — and cuts both strands of the double helix at or near that sequence. The sequence is described as palindromic because the base sequence on one strand reads the same as the complementary strand read in the opposite direction.

Depending on exactly where within the recognition sequence the enzyme cuts, it produces one of two types of end. If it cuts straight across both strands at the same position, it leaves blunt ends. More commonly in genetic engineering, the enzyme cuts the two strands at slightly offset positions, leaving short single-stranded overhangs called sticky ends. These sticky ends are particularly useful because they can form hydrogen bonds with complementary sticky ends on another DNA fragment cut by the same enzyme — making ligation into a vector much more efficient.

To isolate a specific gene using restriction enzymes, the same enzyme must be used to cut both the donor DNA (containing the gene) and the vector DNA (such as a plasmid). This ensures that the sticky ends on the gene fragment are complementary to the sticky ends on the opened vector, allowing the two to anneal and be joined by DNA ligase.

One limitation of this method is that the desired gene must be flanked by appropriate restriction enzyme recognition sequences. If the gene itself contains the same recognition sequence internally, the enzyme will cut within the gene and destroy it. Careful selection of the restriction enzyme — one whose recognition sequence does not appear within the gene — is therefore essential.

4. Method 3 — Creating a Gene Artificially Using a Gene Machine

The third method removes the need for a biological source entirely. If the base sequence of the desired gene is already known — for example, from genome sequencing data — a gene machine can synthesise the gene chemically, one nucleotide at a time, in an automated process.

The gene machine builds the DNA strand through a series of chemical reactions, adding nucleotides in the correct order according to the programmed sequence. Short oligonucleotide sections are synthesised separately and then joined together to produce the full-length gene. The process is entirely in vitro — no cells are required at any stage.

Because the sequence is programmed directly, the gene machine can also be used to introduce deliberate modifications — for example, optimising codon usage for expression in a particular host organism, or adding regulatory sequences. This level of control is not possible with the other two methods, which are constrained by the sequence of the biological source material.

5. Comparing the Three Methods

Now that all three methods are clear, it is worth comparing them directly. Examiners frequently ask you to evaluate which method is most appropriate for a given scenario, so understanding the conditions that favour each one is essential.

Notice that both cDNA and the gene machine produce intron-free DNA, making them the preferred choices when the gene is to be expressed in a prokaryotic host. Restriction enzyme digestion produces a fragment that includes introns, which limits its use to eukaryotic expression systems unless the introns are subsequently removed.

6. Exam Technique and Common Mistakes

Questions on this topic tend to fall into two categories: process description questions (describe or explain how a method works) and evaluation questions (suggest or evaluate which method is most appropriate). Each demands a different approach.

For process description questions, examiners award marks for specific mechanistic detail. Saying 'reverse transcriptase makes DNA from RNA' will earn one mark at most. Saying 'reverse transcriptase catalyses the synthesis of a complementary DNA strand from the mRNA template using free DNA nucleotides, following complementary base pairing rules' is worth considerably more. Precision in enzyme names, direction of synthesis, and the role of base pairing are all mark-winning details.

For evaluation questions, structure your answer around the specific context given. If the question states the gene will be expressed in bacteria, immediately flag that introns are a problem — this signals to the examiner that you understand the eukaryote–prokaryote distinction. Then justify your chosen method with reference to that specific constraint.