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

Section 1 of 27Recombinant 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.

Section 2 of 27Steps 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.

Section 3 of 27Enzymes in genetic engineering

Enzyme	Role
Restriction endonuclease	Cuts DNA at a specific base sequence to isolate the gene and to open the vector, leaving sticky ends
Reverse transcriptase	Makes single-stranded cDNA from mRNA
DNA polymerase	Converts single-stranded cDNA to double-stranded DNA; used in PCR
DNA ligase	Forms phosphodiester bonds to splice the gene into the vector

Section 4 of 27Vectors, 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.

Section 5 of 27Gene 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.

Section 6 of 27Polymerase 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.

Section 7 of 27Gel 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.

Section 8 of 27Microarrays 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.

Section 9 of 27The enzyme toolkit

Enzyme	Job
Restriction endonuclease	Cuts DNA at specific base sequences to isolate a gene or open a vector
Reverse transcriptase	Makes single-stranded cDNA from an mRNA template
DNA polymerase	Converts single-stranded cDNA to double-stranded DNA by pairing free nucleotides, as in replication
DNA ligase	Forms phosphodiester bonds in the sugar-phosphate backbone to join the gene into the vector

Section 10 of 27Why start from mRNA?

Why start from mRNA?

It is easier to find mRNA with the right characteristics than to locate a gene in the whole genome.

• Specialised cells make specific mRNA, e.g. pancreatic beta-cells contain large amounts of insulin mRNA.

Mature mRNA contains no introns, so the cDNA is a continuous coding sequence a bacterium can read.

Route: mRNA → (reverse transcriptase) single-stranded cDNA → (DNA polymerase) double-stranded gene → (ligase) inserted into vector.

Section 11 of 27Vectors

Vector

• Vector carries the desired gene into a foreign host cell.

The commonest vector is a plasmid, a small circular ring of double-stranded DNA.

Viruses and liposomes (vesicles with a phospholipid layer) can also be used.

Preparing a plasmid vector needs restriction endonuclease and DNA ligase.

The same restriction endonuclease must open the plasmid as cut out the gene, so the ends are complementary sticky ends.

Section 12 of 27Promoters

Promoter

• Promoter the region of DNA that determines which gene is expressed, as the site where RNA polymerase binds to begin transcription.

It is a length of non-coding DNA with a specific function.

Promoters are included in plasmids engineered to make human insulin.

Without a promoter the inserted gene would not be transcribed.

Section 13 of 27Marker genes

Marker gene

• Marker gene transferred with the desired gene to show which cells have been successfully altered and contain recombinant DNA.

The plasmid carries the desired gene, a promoter and the marker.

Antibiotic resistance genes were commonly used.

• Bacteria are grown on agar containing that antibiotic and only cells that took up the plasmid survive.

Green fluorescent protein (GFP) makes modified cells glow under suitable light.

• It is quick, non-destructive and avoids spreading antibiotic resistance.

Section 14 of 27Gene editing

Gene (genome) editing

• Gene (genome) editing inserting, deleting or replacing DNA at specific sites known to cause disease.

It is genetic engineering without introducing foreign DNA into the genome.

It manipulates the genome much more accurately than older vector methods.

Section 15 of 27Polymerase chain reaction (PCR)

1

PCR is an in vitro method of DNA amplification.

2

It is used for DNA profiling (identifying criminals, paternity) and genetic engineering.

3

It produces large quantities of DNA or RNA fragments from very small quantities, even a single molecule.

4

The mixture needs template DNA, primers, free nucleotides and heat-stable Taq polymerase.

Section 16 of 27Stages of each PCR cycle

Stage	Temperature	What happens
Denaturation	About 95 °C	Hydrogen bonds break and the DNA separates into single strands
Annealing	About 55-60 °C	Primers bind to complementary sequences on each strand
Elongation	72 °C for at least a minute	Taq polymerase builds complementary strands to produce new identical double-stranded DNA

Section 17 of 27Taq polymerase

Taq polymerase

It comes from a thermophilic bacterium, so it is not denatured at the 95 °C stage.

72 °C is its optimum temperature.

Each cycle doubles the amount of DNA.

Section 18 of 27Principle of gel electrophoresis

Gel electrophoresis

• Gel electrophoresis analyses DNA, RNA and proteins by separating them according to size (mass) and net charge.

Positively charged molecules move to the cathode (negative pole); negatively charged molecules move to the anode (positive pole).

DNA is negatively charged because of its phosphate groups, so it moves to the anode.

Smaller fragments move faster and further through the gel.

Section 19 of 27Method

1

Make an agarose gel plate in a tank by pouring liquid agarose and letting it set.

2

Mould wells into one end of the gel with a comb as it solidifies.

3

Submerge the gel in an electrolyte solution.

4

Load the DNA fragments into the wells with a micropipette.

5

Switch on the current so the fragments separate into bands.

Section 20 of 27Reading a gel

1

In DNA profiling VNTRs (variable number tandem repeats), unique to each person except identical twins, are separated.

2

Band positions are compared with a ladder of known fragment lengths.

• The distance moved is inversely related to the logarithm of fragment size.

3

Two samples with the same band pattern at several VNTR loci very likely come from the same person.

4

In paternity testing each band in the child must appear in the mother or father.

Section 21 of 27DNA microarrays

Microarrays

• Microarrays detect the expression of thousands of genes at once and identify genes present in a genome.

Use in pathology: comparing healthy and diseased cells.

Active genes make many mRNA copies by transcription, and then proteins by translation.

Fluorescent tags of different colours (red, green, yellow) show which genes are present and active.

A spot with no colour shows the gene is absent in both samples.

Section 22 of 27Reading microarray results

Reading microarray results

Sample mRNA is converted to labelled cDNA, which hybridises only to the complementary probe spot.

Spot position identifies the gene.

Brightness shows the amount of expression: more light means more mRNA and a more active gene.

Section 23 of 27What bioinformatics means

Bioinformatics

• Bioinformatics combines biology, computer science and statistics to collect, store, organise and analyse biological data.

Gene sequencing and microarrays produce far more data than can be compared by hand.

Datasets include genome and gene sequences, gene expression patterns and protein amino acid sequences and functions.

Researchers use searchable online databases and analysis software to find patterns.

Section 24 of 27Databases and searches

1

A nucleotide sequence database stores DNA or RNA sequences.

2

A microarray database records which genes are expressed and the amount of mRNA in different cells.

3

Protein databases hold protein sequence and structure information.

4

BLAST compares a query sequence with stored sequences and finds similar regions.

• Similarity can suggest a related gene, a possible protein function or an evolutionary relationship.
• It does not prove identical function by itself.

Section 25 of 27Why comparisons matter

1

After sequencing a genome, scientists compare it with other genomes to estimate how closely organisms are related.

2

Conserved sequences identify genes worth further study.

3

Comparisons help select a model organism, such as the fruit fly, for human biology experiments.

4

A model is useful depending on which genes and pathways are shared.

Section 26 of 27Key terms

Term	Meaning
Recombinant DNA	DNA containing nucleotides from a different source.
Restriction endonuclease	Enzyme cutting DNA at a specific recognition sequence.
Vector	Carrier used to transfer a gene into a cell, e.g. a plasmid.
Promoter	DNA region where RNA polymerase binds to start transcription.
PCR	In vitro method of amplifying DNA.
Gel electrophoresis	Separation of molecules by size and charge in a gel.
Microarray	Chip with probes used to detect many genes at once.
Bioinformatics	Analysis of biological data with computers.

Term	Meaning
Restriction endonuclease	Enzyme cutting DNA at a specific sequence.
Reverse transcriptase	Enzyme making DNA from mRNA.
DNA ligase	Enzyme joining DNA backbones.
Vector	Carrier delivering a gene into a host cell.
Promoter	Site where RNA polymerase binds to start transcription.
cDNA	DNA copy of mRNA without introns.

Term	Meaning
Marker gene	Gene used to identify cells containing recombinant DNA.
GFP	Green fluorescent protein used as a marker.
Gene editing	Changing DNA at a specific site without adding foreign DNA.
PCR	In vitro amplification of DNA.
Primer	Short DNA sequence that binds to the target to start copying.
Taq polymerase	Heat-stable DNA polymerase.

Term	Meaning
Gel electrophoresis	Separation of molecules by size and charge in a gel.
VNTR	Variable number tandem repeat; sequence repeated a different number of times in different people.
Anode	Positive electrode.
Microarray	Chip carrying probes to detect genes and their expression.
Ladder	Mixture of fragments of known length used to size others.

Term	Meaning
Bioinformatics	Analysis of biological data using computers and statistics.
BLAST	Tool that finds similar sequences in databases.
Database	Searchable store of biological data.
Conserved sequence	Sequence that stays similar between species.
Model organism	Organism used in research to understand another.

Section 27 of 27Exam technique

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.

1

Saying ligase cuts DNA — restriction enzymes cut and ligase joins.

2

Using different restriction enzymes for gene and plasmid.

3

Forgetting the promoter — without it the gene is not expressed.

4

Saying cDNA contains introns.

1

Learning only one marker method — know both antibiotic resistance and fluorescence, and why fluorescence is preferred.

2

Saying gene editing introduces foreign DNA.

3

Mixing up PCR temperatures.

4

Saying Taq polymerase is denatured at 95 °C.

1

Saying DNA moves to the cathode — it moves to the anode.

2

Saying larger fragments move further.

3

Saying the colour of a spot shows level of expression — brightness shows level.

4

Forgetting the wells are at the negative end.

1

Saying a BLAST match proves the same function.

2

Saying bioinformatics only stores data — it also analyses it.

3

Forgetting that more similar sequences mean more closely related organisms.

4

Describing a model organism without saying which genes or pathways are shared.