Scientists use many different techniques in genetic engineering.
(a) Sometimes the gene for genetic engineering cannot be extracted from the donor organism. Instead, the gene is synthesised using one of two different methods.
Outline the two methods for synthesising a gene for use in genetic engineering.
(b) DNA ligase and DNA polymerase are two enzymes that are used in genetic engineering.
Complete Table 8.1 to show the roles of DNA ligase and DNA polymerase in genetic engineering.
Use a tick (✓) if the enzyme has the role or a cross (x) if the enzyme does not have the role.
(c) The polymerase chain reaction (PCR) is used to make many copies of a gene.
Three temperatures are used in a PCR cycle.
State the three temperatures that are used, and outline what happens at each temperature during a PCR cycle.
▶️ Answer/Explanation
(a)
The two methods for synthesising a gene are:
- Using reverse transcriptase: This method involves making complementary DNA (cDNA) from messenger RNA (mRNA). The enzyme reverse transcriptase creates a DNA strand from the mRNA template, which is then converted into double-stranded DNA.
- Chemical synthesis: In this method, the gene is synthesized directly by joining nucleotides together in the correct sequence. This is done using known amino acid or nucleotide sequences from databases, and the nucleotides are chemically bonded to form the desired DNA sequence.
(b)
Explanation: DNA ligase is responsible for joining DNA fragments together by forming phosphodiester bonds between the sugar-phosphate backbones. This is essential for inserting genes into plasmids. DNA polymerase, on the other hand, adds free nucleotides to synthesize new DNA strands during processes like PCR, but it doesn’t join separate DNA fragments together.
(c)
The three temperatures used in PCR and their functions are:
- Denaturation (90-98°C): At this high temperature, the hydrogen bonds between the two strands of DNA break, causing the double-stranded DNA to separate into single strands. This provides the template for replication.
- Annealing (50-65°C): The temperature is lowered to allow primers to bind (anneal) to their complementary sequences on the single-stranded DNA templates. Primers are short DNA sequences that mark the starting points for DNA synthesis.
- Extension (68-75°C): At this optimal temperature for Taq polymerase (a heat-stable DNA polymerase), the enzyme synthesizes new DNA strands by adding nucleotides complementary to the template strand, starting from the primers.
These three steps constitute one PCR cycle, which is typically repeated 25-35 times to exponentially amplify the target DNA sequence.