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19.119 · Genetic Technology

Principles of Genetic Technology — Cambridge AS & A Level Biology 9700 Revision Notes & Topical Questions

10 min read 27 parts Lesson 1 of 20 12 quizzes

📚 Cambridge AS & A Level Biology 9700 Hub / Topical Revision
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19.1 Principles of Genetic Technology — CAMBRIDGE AS & A LEVEL BIOLOGY 9700 — REVISION NOTES notes: Describe recombinant DNA, genetic engineering, PCR, gel electrophoresis, microarrays and bioinformatics.The genetic code is universal — almost all organisms use the same four bases…

  • Recombinant DNA (rDNA)
  • Transgenic organism
  • Genetically modified organism (GMO)
  • CRISPR-Cas9
  • Restriction endonuclease
  • Identify the desired gene

Part 1 of 8Recombinant DNA

1The genetic code is universal
  • The genetic code is universal almost all organisms use the same four bases and the same codons for the same amino acids.
  • Genetic information is therefore transferable between species.
2Recombinant DNA (rDNA)
  • DNA with nucleotides introduced from another source, usually another species.
3Transgenic organism
  • Transgenic organism contains nucleotide sequences from a different species.
4Genetically modified organism (GMO)
  • Genetically modified organism (GMO) any organism with introduced genetic material.

Part 2 of 8Steps of genetic engineering

1
Identify the desired gene.
2
Isolate it by cutting from a chromosome with restriction endonucleases, by making cDNA from mRNA with reverse transcriptase,
  • or by artificial synthesis.
3
Multiply the gene using PCR.
4
Transfer it into the organism using a vector (plasmid, virus or liposome).
5
Identify cells that took up the gene using a marker, then clone them.

Part 3 of 8Enzymes in genetic engineering

EnzymeRole
Restriction endonucleaseCuts DNA at a specific base sequence to isolate the gene and to open the vector, leaving sticky ends
Reverse transcriptaseMakes single-stranded cDNA from mRNA
DNA polymeraseConverts single-stranded cDNA to double-stranded DNA; used in PCR
DNA ligaseForms phosphodiester bonds to splice the gene into the vector

Part 4 of 8Vectors, promoters and markers

Plasmids are the commonest vector; they self-replicate; recombinant plasmids enter bacteria by transformation (ice-cold calcium chloride then 40 °C,
  • or electroporation).
Promoter
  • Promoter the site where RNA polymerase binds; an appropriate promoter must be added so the gene is expressed.
Marker genes show which cells carry recombinant DNA.
  • Antibiotic resistance genes risk spreading resistance by conjugation or transduction.
  • GFP fluoresces green under UV light and is easier, cheaper and safer;
    • GUS gives coloured or fluorescent products.

Part 5 of 8Gene editing

Gene editing inserts, deletes or replaces DNA at specific sites without introducing foreign DNA.
CRISPR-Cas9
  • CRISPR-Cas9 a guide RNA leads the Cas9 enzyme to cut DNA at a specific point, then the faulty DNA is replaced.
It is used in gene therapies, e.g. for cystic fibrosis and sickle cell anaemia.

Part 6 of 8Polymerase chain reaction (PCR)

1
PCR amplifies DNA in vitro; 20 cycles give about a million copies, since the DNA doubles each cycle (N = N0 × 2n).
2
Requires target DNA, primers, Taq polymerase, free nucleotides and buffer.
3
Stages per cycle:
  • Denaturation at 95 °C breaks hydrogen bonds.
  • Annealing at 50-60 °C lets primers bind.
  • Elongation at 72 °C lets Taq polymerase build the new strands.
4
Taq polymerase from Thermus aquaticus does not denature at high temperature.

Part 7 of 8Gel electrophoresis

1
Separates DNA, RNA and proteins by size and net charge.
2
DNA is negatively charged (phosphate groups), so it moves towards the anode; shorter fragments move faster and further.
3
Agarose gel is used for DNA and polyacrylamide for proteins.
4
DNA is amplified by PCR, cut by restriction enzymes near VNTRs, loaded into wells and run in an electrolyte.
5
Fragments are made visible by labelled probes (radioactive or fluorescent) and compared with a ladder or control.
6
Proteins are separated using buffers to hold the pH constant, e.g. normal and sickle cell haemoglobin.

Part 8 of 8Microarrays and bioinformatics

Microarray
  • Microarray a small chip with thousands of gene spots holding single-stranded DNA probes.
  • Fluorescent-tagged DNA hybridises to complementary probes;
    • red, green or yellow spots show which species have the gene.
  • To study expression, mRNA is converted to cDNA, tagged and hybridised;
    • light intensity shows the amount of mRNA.
Bioinformatics
  • Bioinformatics the collection, organisation, analysis and storage of large biological data sets using computers and statistics.
  • Databases include EMBL, ArrayExpress and Protein Data Bank in Europe; BLAST finds similar sequences.
  • Comparing genomes shows relatedness and helps select model organisms such as Drosophila.

Key terms

TermMeaning
Recombinant DNADNA containing nucleotides from a different source.
Restriction endonucleaseEnzyme cutting DNA at a specific recognition sequence.
VectorCarrier used to transfer a gene into a cell, e.g. a plasmid.
PromoterDNA region where RNA polymerase binds to start transcription.
PCRIn vitro method of amplifying DNA.
Gel electrophoresisSeparation of molecules by size and charge in a gel.
MicroarrayChip with probes used to detect many genes at once.
BioinformaticsAnalysis of biological data with computers.

Avoid these mistakes

1
Saying cDNA contains introns — it is made from mature mRNA and has none.
2
Saying restriction enzymes cut the gene — they cut the DNA at a recognition sequence.
3
Using different restriction enzymes for the gene and the plasmid — the same enzyme gives complementary sticky ends.
4
Forgetting that DNA moves to the positive pole because it is negatively charged.
5
Mixing up the PCR temperatures and stages.

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