How do we read, copy and engineer DNA, and how is DNA profiling used?
6.1.3 Manipulating genomes: the principles of DNA sequencing, the polymerase chain reaction (PCR) and gel electrophoresis; the use of restriction enzymes and ligase to produce recombinant DNA in genetic engineering; the principles of gene editing; and the use of DNA profiling.
A focused answer to the OCR H420 6.1.3 dot point on manipulating genomes. Covers DNA sequencing, the polymerase chain reaction and gel electrophoresis, restriction enzymes and ligase in genetic engineering, the principles of gene editing, and DNA profiling.
Reviewed by: AI editorial process; not yet individually human-reviewed
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What this dot point is asking
OCR wants you to explain DNA sequencing, the polymerase chain reaction and gel electrophoresis, the use of restriction enzymes and ligase to make recombinant DNA in genetic engineering, the principles of gene editing, and the use of DNA profiling.
The answer
DNA sequencing
DNA sequencing determines the order of bases in a length of DNA. Modern high-throughput (next-generation) methods sequence whole genomes rapidly and cheaply, allowing comparison of genomes between species (for phylogeny) and between individuals (for medicine). Knowing the sequence lets us predict the amino acid sequence and so the protein.
The polymerase chain reaction (PCR)
PCR makes many copies of a DNA fragment in vitro, cycling through three temperatures:
- Denaturation (about 95 degrees Celsius): the high temperature breaks the hydrogen bonds, separating the two DNA strands.
- Annealing (about 55 degrees Celsius): primers bind to the complementary sequences flanking the target region.
- Extension (about 72 degrees Celsius): heat-stable Taq polymerase adds complementary nucleotides from the primers, building new strands.
Each cycle doubles the DNA, giving exponential amplification, so a tiny sample can be amplified for analysis.
Gel electrophoresis
Gel electrophoresis separates DNA fragments by size. DNA samples are placed in wells in an agarose gel and an electric field is applied; because DNA is negatively charged (its phosphate groups), the fragments move towards the positive electrode. Smaller fragments move faster and further through the gel mesh, so the fragments separate into bands by length. It is used in sequencing and DNA profiling.
Genetic engineering and recombinant DNA
To insert a gene (for example to make bacteria produce human insulin):
- The desired gene is obtained (cut from DNA, or made from mRNA using reverse transcriptase, or synthesised).
- A restriction enzyme cuts the gene and the plasmid vector at the same recognition sequence, leaving complementary sticky ends.
- The gene and plasmid are mixed; the sticky ends base-pair, and DNA ligase joins the backbones, forming a recombinant plasmid.
- The plasmid is taken up by a host (such as a bacterium), which expresses the gene. Marker genes identify the cells that took up the plasmid.
Gene editing
Gene editing (for example using CRISPR-Cas9) allows precise changes to a specific DNA sequence: a guide molecule targets the enzyme to an exact site, where it cuts the DNA so a base sequence can be removed, corrected or added. It is far more precise than older methods and has potential to correct disease-causing mutations.
DNA profiling
DNA profiling identifies individuals from the variable, non-coding regions of their DNA (short tandem repeats, which vary in number between people). The steps are: extract the DNA, amplify the repeat regions by PCR, cut or separate them, and run gel electrophoresis to produce a pattern of bands (the profile). The chance of two unrelated people sharing a profile is tiny, so it is used in forensics, paternity testing and studying relationships.
Examples in context
Example 1. Bacterial insulin. The human insulin gene is inserted into a bacterial plasmid using restriction enzymes and ligase; the transformed bacteria are cultured to produce human insulin for people with diabetes, a landmark use of recombinant DNA.
Example 2. Forensic DNA profiling. Tiny crime-scene samples are amplified by PCR and profiled by electrophoresis, allowing suspects to be identified or excluded, and have exonerated wrongly convicted people.
Try this
Q1. State the three temperature stages of PCR and what happens at each. [3 marks]
- Cue. Denaturation (about 95 degrees, strands separate), annealing (about 55 degrees, primers bind), extension (about 72 degrees, Taq polymerase builds new strands).
Q2. Explain why smaller DNA fragments travel further in gel electrophoresis. [2 marks]
- Cue. DNA is negatively charged and moves towards the positive electrode; smaller fragments move more easily through the gel mesh, so they travel faster and further than larger fragments.
Q3. Name the enzyme used to join the sugar-phosphate backbones when making recombinant DNA. [1 mark]
- Cue. DNA ligase.
Exam-style practice questions
Practice questions written in the style of OCR exam questions on this dot point, with worked answer explainers. The year tag is the paper they imitate, not the source.
OCR H420/02 20196 marksDescribe how the polymerase chain reaction (PCR) is used to make many copies of a DNA fragment, explaining the role of temperature at each step.Show worked answer →
Take the three temperature stages and what happens at each, then the cycling.
The DNA, primers, free nucleotides and Taq polymerase are mixed. Denaturation (about 95 degrees Celsius): the high temperature breaks the hydrogen bonds between the two strands, separating them.
Annealing (about 55 degrees Celsius): the mixture is cooled so the primers bind (anneal) to their complementary sequences at the ends of the target region.
Extension (about 72 degrees Celsius): Taq polymerase (heat-stable) adds complementary nucleotides to each template strand from the primers, building new strands.
Each cycle doubles the number of copies, so repeating the cycle gives exponential amplification. Markers reward the three named stages with temperatures, the role of primers and Taq polymerase, and the doubling each cycle.
OCR H420/02 20215 marksDescribe how restriction enzymes and ligase are used to insert a human gene into a bacterial plasmid to produce recombinant DNA.Show worked answer →
Cut, match the sticky ends, then join.
A restriction enzyme cuts the DNA at a specific recognition sequence, leaving short single-stranded sticky ends. The same restriction enzyme is used to cut both the human gene out and to open the bacterial plasmid, so they have complementary sticky ends.
The gene and the cut plasmid are mixed; the complementary sticky ends pair by hydrogen bonding (base pairing). DNA ligase then joins the sugar-phosphate backbones (forming phosphodiester bonds), producing a recombinant plasmid containing the human gene.
The plasmid is taken up by the bacterium (a vector), which then expresses the gene. Markers reward the same restriction enzyme giving complementary sticky ends, base pairing, and ligase joining the backbones to form recombinant DNA.
Related dot points
- 6.1.4 Cloning and biotechnology: natural and artificial cloning of plants (including micropropagation and tissue culture) and animals; the use of microorganisms in biotechnology and the conditions in an industrial fermenter; the principles and advantages of using immobilised enzymes; and the asepsis and growth curve of a microbial culture.
A focused answer to the OCR H420 6.1.4 dot point on cloning and biotechnology. Covers natural and artificial cloning of plants and animals, micropropagation and tissue culture, the use of microorganisms and the conditions in an industrial fermenter, immobilised enzymes, and the microbial growth curve.
- 6.1.1 Cellular control: the nature of gene mutations and their effects on proteins; the control of gene expression at the transcriptional level, including operons (the lac operon) and transcription factors; the role of homeobox (Hox) genes in body plan development; and the role of apoptosis (programmed cell death).
A focused answer to the OCR H420 6.1.1 dot point on cellular control. Covers gene mutations and their effects, the control of transcription by the lac operon and transcription factors, the role of homeobox (Hox) genes in body plan development, and apoptosis.
- 2.1.3 Nucleotides and nucleic acids: the semi-conservative replication of DNA and the roles of DNA helicase, DNA polymerase and the complementary base pairing rule; the nature of the genetic code as a triplet code that is degenerate and non-overlapping; the roles of mRNA and tRNA in protein synthesis.
A focused answer to the OCR H420 2.1.3 dot point on DNA replication and the genetic code. Covers semi-conservative replication, the roles of DNA helicase and DNA polymerase, the Meselson-Stahl evidence, and the triplet, degenerate, non-overlapping code with transcription and translation.
- 6.1.2 Patterns of inheritance: monohybrid and dihybrid crosses; the inheritance of codominant and multiple alleles, sex linkage and epistasis; the use of genetic diagrams to predict phenotypic ratios; and the chi-squared test to compare observed and expected results.
A focused answer to the OCR H420 6.1.2 dot point on patterns of inheritance. Covers monohybrid and dihybrid crosses, codominance and multiple alleles, sex linkage and epistasis, genetic diagrams and phenotypic ratios, and the chi-squared test.
- 6.1.2 Populations and evolution: the meaning of a gene pool and allele frequency; the use of the Hardy-Weinberg principle to calculate allele and genotype frequencies; the factors that change allele frequencies (natural selection, genetic drift, the founder effect and migration); and the process of speciation (allopatric and sympatric).
A focused answer to the OCR H420 6.1.2 dot point on populations and evolution. Covers gene pools and allele frequency, the Hardy-Weinberg principle and its calculations, the factors that change allele frequencies including genetic drift and the founder effect, and allopatric and sympatric speciation.
Sources & how we know this
- OCR A Level Biology A (H420) Specification — OCR (2023)