Gene Technologies (A Level Only)

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Recombinant DNA Technology

The genetic code is universal: almost every organism uses the same four bases (A, T, C, G), and the same codons code for the same amino acids in all living things.
Because the code is universal, genetic information is transferable between species, allowing scientists to combine lengths of nucleotides from different sources.
DNA that has been altered by introducing nucleotides from another source is called recombinant DNA (rDNA).
An organism containing nucleotide sequences from a different species is a transgenic organism; any organism with introduced genetic material is a genetically modified organism (GMO).
The mechanisms of transcription and translation are also universal, so transferred DNA can be translated within the cells of the genetically modified organism.
Genetic engineering requires: enzymes (restriction endonucleases, ligase, reverse transcriptase), vectors (e.g. plasmids, viruses, liposomes) to deliver DNA fragments, and markers (genes coding for identifiable substances, e.g. GFP or GUS).
The main steps are: identification and isolation of the desired DNA fragment, multiplication (using PCR), transfer into the organism using a vector, then identification of cells with the new DNA fragment using a marker, which are then cloned.

Producing Fragments of DNA

A desired gene can be obtained by: extraction from donor DNA using restriction endonucleases, synthesis of cDNA from mRNA using reverse transcriptase, or artificial synthesis in a gene machine.
Restriction endonucleases are enzymes found in bacteria that act as a defence against bacteriophages by cutting viral genetic material at specific nucleotide sequences.
Each restriction endonuclease binds to a specific restriction site (specific base sequence), e.g. HindIII always binds to AAGCTT.
They cut the sugar-phosphate backbone either unevenly to give sticky ends (one strand longer than the other) or straight across to give blunt ends.
Sticky ends make it easier to insert the desired gene into another organism's DNA because they can form hydrogen bonds with complementary base sequences on other DNA pieces cut with the same enzyme.
Reverse transcriptase, sourced from retroviruses, uses mRNA as a template to synthesise a single strand of complementary DNA (cDNA); DNA polymerase then converts it into a double-stranded DNA molecule.
Using mRNA is advantageous because specialised cells make very specific types of mRNA (e.g. β-cells produce many insulin mRNAs) and the mRNA (and therefore the cDNA) does not contain introns.
In a gene machine, computers generate the nucleotide sequence from knowledge of the required amino acids; short DNA fragments are produced, joined into longer sequences, and inserted into vectors.

Investigating the Specificity of Restriction Enzymes

The specificity of restriction enzymes can be investigated using extracted DNA and gel electrophoresis.
DNA fragments produced by restriction enzymes are called restriction fragments; digesting a DNA sample with a restriction enzyme produces a mixture of fragments of varying lengths.
In gel electrophoresis, DNA is negatively charged due to its phosphate groups, so it migrates towards the positive electrode when an electric current is applied.
Smaller fragments move faster and farther through the tiny pores of the gel, while larger fragments move more slowly, separating the fragments by size.
DNA is colourless, so bands must be visualised using a stain, a radioactive marker, or fluorescent probes that bind to the DNA.
Because restriction enzymes are highly specific, the same initial DNA sample always produces the same number and sizes of restriction fragments.
An unknown restriction enzyme can be identified by comparing the restriction fragments it produces to those of a known restriction enzyme — the two sets of bands should be the same.

Polymerase Chain Reaction

The polymerase chain reaction (PCR) is the in vitro method of DNA amplification, used to produce large quantities of specific DNA or RNA fragments from very small quantities.
Each PCR reaction requires: target DNA/RNA, primers (forward and reverse), DNA polymerase (usually Taq polymerase), free nucleotides, and a buffer solution to provide the optimum pH.
Primers are short sequences of single-stranded DNA with base sequences complementary to the 3' end of the DNA being copied; they define the region to be amplified and show DNA polymerase where to begin.
Taq polymerase is used because it is thermostable — it does not denature at the high temperatures involved, and its optimum temperature is high enough to prevent annealing of uncopied DNA strands.
Stage 1 – Denaturation: DNA is heated to 95°C, breaking the hydrogen bonds between the two DNA strands.
Stage 2 – Annealing: the temperature is decreased to 50–60°C so that primers can anneal to the ends of the single strands.
Stage 3 – Elongation/Extension: the temperature is increased to 72°C for at least a minute, the optimum temperature for Taq polymerase to build complementary strands.
In each cycle the DNA is doubled, so a standard run of 20 cycles produces a million DNA molecules.

Culture of Transformed Host Cells

Gene cloning can be carried out in vivo using bacteria, which are the most common host cells because they increase in numbers rapidly and are relatively easy to culture.
Step 1: A DNA fragment is isolated (by restriction endonucleases, reverse transcriptase, or a gene machine), and promoter and terminator regions are added to ensure replication.
Step 2: The DNA fragments are inserted into vectors (commonly plasmids) using restriction endonucleases and ligase enzymes.
Step 3: The vectors are transported into bacterial host cells; cells containing the modified plasmids are called transformed host cells.
Step 4: The bacteria multiply rapidly under optimum conditions.
Step 5: Marker genes are used to identify successfully transformed bacteria — only a small fraction take up the plasmid, and those that have not taken up the desired gene are destroyed.
Step 6: The remaining bacteria are cultured, and every time a bacterium divides, the desired gene is cloned.
Using this in vivo method, recombinant DNA can be used to produce recombinant proteins such as insulin.

Uses of Recombinant DNA Technology

Recombinant proteins are generated using microorganisms such as bacteria, yeast, or animal cells in culture, and are used for research and treatments (e.g. diabetes, cancer, infectious diseases, haemophilia).
Most recombinant human proteins are produced using eukaryotic cells (e.g. yeast or animal cells) because they carry out the post-translational modification required to produce a suitable human protein.
Advantages of using genetically engineered organisms to produce recombinant human proteins include: more cost-effective large volumes, simpler and faster production, reliable supply, and proteins identical to human proteins.
Insulin was the first recombinant human protein approved for diabetes treatment: plasmids are cut with restriction endonucleases, human DNA is spliced in with DNA ligase, and the recombinant plasmids are inserted into×Escherichia coli×by transformation.
The transgenic bacteria are identified by markers, isolated, purified, placed into fermenters, multiply by binary fission, and express human insulin, which is extracted and purified.
Advantages of recombinant insulin include: identical to human insulin, reliable supply, fewer ethical/moral/religious concerns, fewer rejection problems or allergic reactions, and cheaper large-volume production.
Factor VIII, a blood-clotting protein that haemophiliacs cannot produce, is made by genetically modified hamster kidney and ovary cells; advantages include fewer ethical concerns, less risk of transmitting infection (e.g. HIV), and a greater production rate.
Gene therapy alters a person's genetic material to treat or cure diseases, and is used to introduce corrected copies of genes for conditions such as cystic fibrosis, haemophilia, and severe combined immunodeficiency (SCID).

Gene Therapy and Genetic Engineering in Agriculture

All current gene therapies target somatic (body) cells, so changes are not inherited by future generations; however, the effects are often short-lived.
Ex vivo gene therapy inserts the new gene via a virus vector into cells outside the body (e.g. blood or bone marrow cells are extracted, exposed to the virus, grown in the laboratory, and returned by injection).
In vivo gene therapy inserts the new gene via a vector into cells inside the body.
Inserting new genetic material into germ cells is illegal in humans because changes would be potentially permanent and could be inherited by future generations.
SCID is caused by a lack of adenosine deaminase (ADA); ex vivo somatic gene therapy uses a virus to transfer a normal ADA allele into T-lymphocytes, but it is not a permanent cure as T-lymphocytes are replaced over time.
Social and ethical considerations of gene therapy include: potential side effects (e.g. leukaemia-like cancer after retroviral therapy), whether germline therapy should be allowed, commercial viability, expense, and the right to determine which genes can be altered.
Crop plants have been genetically modified to be resistant to herbicides and pests (increasing yield) and enriched in vitamins; farmed animals have been modified to grow faster, though this is rarer due to ethical concerns.
Benefits of genetic engineering over selective breeding include: organisms with desired characteristics are produced more quickly, all organisms contain the desired characteristic, and the characteristic may come from a different species or kingdom.

DNA Probes & DNA Hybridisation

A DNA probe is a short length of single-stranded DNA with a known base sequence complementary to the specific base sequence of a known allele, usually attached to a radioactive or fluorescent label.
DNA hybridisation is the process where two complementary single-stranded DNA molecules combine through base pairing to form a single double-stranded DNA molecule.
To use DNA probes to locate specific alleles: a cell sample is taken (blood, cheek swab, umbilical cord, or amniotic fluid), DNA is extracted and purified, then amplified using PCR.
The amplified DNA is digested with restriction endonucleases because whole DNA molecules are too long to analyse in one go, and the fragments are separated by gel electrophoresis.
The DNA bands are transferred to a nylon membrane and made single-stranded by breaking the hydrogen bonds between complementary base pairs.
Labelled DNA probes are added; they anneal to any complementary DNA fragments present, and the membrane is washed to remove excess probes.
For fluorescent labels, UV light detects their position; for radioactive labels, autoradiography is used.
If the label shows up on a restriction fragment, the DNA at that position is from the harmful allele; if no labels show up, the test DNA does not contain that harmful allele — though the test often only detects one specific harmful allele.

Screening Patients

Genetic screening is the testing of an embryo, fetus, or adult to analyse their DNA and identify individuals carrying an allele for a particular disorder.
DNA samples can be obtained by taking tissue samples from adults or embryos produced by IVF, or by chorionic villus sampling or amniocentesis of embryos and fetuses in the uterus.
BRCA1 and BRCA2 are genes that produce tumour suppressor proteins regulating cell growth; faulty alleles increase the risk of breast and ovarian cancers and can be inherited from either parent.
Advantages of screening for BRCA mutations include: the person may take preventative measures (e.g. elective mastectomy), screening can begin earlier or more frequently, and it enables participation in research and clinical trials.
Advantages of genetic screening generally: people can make sensible lifestyle choices to reduce disease risk, potential parents can choose whether to have biological children, and people can participate in research and clinical trials.
Disadvantages include: nothing positive may be done with the information (leading to depression or fear), higher life insurance prices, fear of genetic discrimination, and pressure on parents not to have children.
Screening may be used to look more broadly at a potential child's genetic make-up, raising ethical questions about 'designer babies'; some religions consider such interference with reproduction highly immoral.

Genetic Counselling & Personalised Medicine

Genetic counsellors help individuals understand and process the results of genetic screening, reading out results and explaining what they mean.
Counsellors may also be seen before screening to inform an individual of possible results.
They may discuss: the chances of developing an inherited disease, lifestyle changes to reduce or manage risk, therapeutic treatments possible, the chance of having a child with a certain disease, termination of pregnancy, financial implications, and ethical issues.
Personalised medicine involves developing more targeted drugs to treat a variety of human diseases, as well as developing synthetic tissues.
Information from genome projects like the Human Genome Project (HGP) can be used to develop genomic medicine, which uses information about an individual's genes to influence their clinical care.
Genetic screening allows individuals with a high chance of developing specific diseases to be identified so that preventative measures or precautions can be taken.

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1.What is meant by the term recombinant DNA?
Easy
- ADNA that contains nucleotide sequences from more than one source or species
- BDNA that has been copied many times by the polymerase chain reaction
- CDNA that has been cut into fragments by restriction endonucleases
- DDNA that is made entirely from mRNA using reverse transcriptase
2.Which enzyme is used to join the sugar-phosphate backbone of a DNA fragment to a plasmid vector?
Easy
- ADNA ligase
- BRestriction endonuclease
- CReverse transcriptase
- DDNA polymerase
3.A DNA probe is a short, single-stranded piece of DNA. Which statement best describes its base sequence?
Easy
- AIt is complementary to the specific base sequence of a known allele
- BIt is identical to the base sequence of the target allele
- CIt is complementary to the base sequence of any allele at the locus
- DIt is a random sequence that binds to all DNA fragments
4.During the polymerase chain reaction, the temperature is lowered to between 50 °C and 60 °C. What is the purpose of this step?
Medium
- ATo allow primers to anneal to the single-stranded DNA
- BTo break the hydrogen bonds between the two DNA strands
- CTo provide the optimum temperature for Taq polymerase to build new strands
- DTo activate the buffer solution so that it maintains the optimum pH
5.A sample of DNA was cut with a restriction enzyme and the fragments were separated by gel electrophoresis. The diagram shows the positions of the bands obtained. Which conclusion is best supported by the results?
Medium
- AThe DNA sample contained several fragments of different lengths
- BThe restriction enzyme cut the DNA at random sites
- CAll the DNA fragments had the same net charge
- DThe DNA fragments moved towards the negative electrode
6.In genetic fingerprinting, why are variable number tandem repeats (VNTRs) useful?
Medium
- AThe number of repeats varies between individuals, producing unique banding patterns
- BThey code for proteins that differ between individuals
- CThey are the same length in all individuals, so they can be used as a standard
- DThey are found only in the coding regions of DNA
7.Which of the following are required for the polymerase chain reaction? (Select all that apply.)
Medium
- ATarget DNA
- BPrimers
- CTaq polymerase
- DFree nucleotides
- ERestriction endonucleases
8.Which of the following statements about genetic screening are correct? (Select all that apply.)
Hard
- AIt can identify individuals carrying an allele for a particular disorder
- BIt may enable people to make lifestyle choices that reduce the risk of disease
- CIt always provides a definite cure for the genetic disorder
- DIt may lead to higher life insurance premiums
- EIt can allow potential parents to make informed reproductive decisions

Plus de questions

9.The genetic code is universal, meaning that the same codons code for the same amino acids in almost all living organisms.
Easy
True or false?
10.Restriction endonucleases cut DNA at random sites along the molecule.
Easy
True or false?
11.During gel electrophoresis, DNA fragments move towards the negative electrode because DNA is positively charged.
Easy
True or false?
12.Taq polymerase is used in PCR because it is thermostable and does not denature at the high temperatures used in the reaction.
Medium
True or false?
13.DNA hybridisation is the process by which two complementary single-stranded DNA molecules combine through base pairing.
Medium
True or false?
14.Match each term with its correct description.
Medium
- Restriction endonuclease
- DNA ligase
- Reverse transcriptase
- DNA probe
- Joins DNA fragments by forming sugar-phosphate bonds
- Cuts DNA at specific recognition sites
- Short single-stranded DNA complementary to a known allele
- Synthesises cDNA from an mRNA template
15.Match each stage of PCR with the temperature at which it occurs.
Medium
- Denaturation
- Annealing
- Elongation
- 95 °C
- 50–60 °C
- 72 °C
16.Place the stages of in vivo gene cloning using bacteria into the correct order.
Medium
- A DNA fragment is isolated
- The DNA fragment is inserted into a vector
- The vector is transported into bacterial host cells
- Marker genes are used to identify transformed bacteria
- The transformed bacteria are cultured
17.Place the steps of using a DNA probe to locate a specific allele into the correct order.
Medium
- A cell sample is taken from the patient
- DNA is extracted and purified
- The DNA is amplified using PCR
- Restriction endonucleases digest the DNA
- Fragments are separated by gel electrophoresis
- The bands are transferred to a nylon membrane
- Labelled DNA probes are added to the membrane
18.Why do the DNA fragments move through the gel during electrophoresis?
Medium
- ADNA is negatively charged and moves towards the positive electrode
- BDNA is positively charged and moves towards the negative electrode
- CDNA is neutral and moves randomly through the gel
- DDNA is attracted to the negative electrode by the phosphate groups
19.Why do the DNA fragments appear as bands at different positions in the gel?
Medium
- ASmaller fragments move faster and further through the gel than larger fragments
- BLarger fragments move faster and further through the gel than smaller fragments
- CAll fragments move at the same speed but different distances
- DFragments move at different speeds depending on their charge only
20.Restriction enzymes are highly specific, so the same enzyme will always produce the same number and sizes of restriction fragments from the same DNA sample.
Easy
True or false?
21.Which of the following are needed by genetic engineers to modify an organism? (Select all that apply.)
Medium
- AEnzymes such as restriction endonucleases and ligase
- BVectors such as plasmids and viruses
- CMarkers such as GFP
- DAntibiotics to kill all bacteria
- EA thermocycler to keep cells at 4 °C
22.Match each method of producing DNA fragments with the starting material it uses.
Medium
- Restriction endonucleases
- Reverse transcriptase
- Gene machine
- mRNA
- Nucleotides only (no template)
- Extracted DNA
23.Place the steps of genetic engineering an organism into the correct order.
Medium
- Identification of the DNA fragment or gene
- Isolation of the desired DNA fragment
- Multiplication of the DNA fragment using PCR
- Transfer into the organism using a vector
- Identification and cloning of cells with the new DNA fragment
24.Which of the following statements about sticky ends are correct? (Select all that apply.)
Medium
- ASticky ends are produced when a restriction enzyme cuts the sugar-phosphate backbone unevenly.
- BSticky ends allow the DNA fragment to form hydrogen bonds with complementary base sequences on other DNA cut with the same enzyme.
- CSticky ends are produced when a restriction enzyme cuts straight across both strands to give blunt ends.
- DSticky ends result in one strand of the DNA fragment being longer than the other.
- ESticky ends are formed by DNA ligase joining two blunt-ended fragments.
25.Which of the following is an advantage of using reverse transcriptase to isolate a desired gene compared with using restriction endonucleases?
Medium
- AThe cDNA produced does not contain introns.
- BThe cDNA is double-stranded immediately after reverse transcription.
- CRestriction endonucleases cannot cut DNA.
- DThe mRNA used as a template is double-stranded.
26.Which of the following are required in a polymerase chain reaction? (Select all that apply.)
Medium
- ADNA polymerase
- BPrimers
- CFree nucleotides
- DBuffer solution
- ERestriction endonucleases
27.Match each component of PCR with its function.
Easy
- Primers
- Taq polymerase
- Free nucleotides
- Buffer solution
- Provide optimum pH
- Define the region to be amplified and provide a starting point for DNA polymerase
- Build the new DNA strands
- Used in the construction of new DNA strands
28.In PCR, the annealing stage occurs at 72 °C.
Easy
True or false?
29.Place the stages of a single PCR cycle in the correct order.
Medium
- Denaturation
- Annealing
- Elongation
30.During PCR, why is the temperature decreased to between 50 °C and 60 °C?
Medium
- ATo allow primers to anneal to the single-stranded DNA.
- BTo break the hydrogen bonds between the DNA strands.
- CTo allow Taq polymerase to build the new DNA strands.
- DTo activate the buffer solution.
31.Which of the following are advantages of using recombinant human proteins such as insulin compared with proteins extracted from animals? (Select all that apply.)
Medium
- AThere is a reliable supply available.
- BThere are fewer ethical, moral or religious concerns.
- CThere are fewer rejection problems and allergic reactions.
- DThe protein is always extracted from human blood.
- EThere is a risk of transmitting animal diseases.
32.Which of the following is an advantage of using eukaryotic cells rather than prokaryotic cells to produce recombinant human proteins?
Medium
- AEukaryotic cells carry out post-translational modification.
- BEukaryotic cells are always simpler to culture.
- CEukaryotic cells do not require a vector.
- DEukaryotic cells produce proteins without transcription.
33.Which of the following are advantages of genetic screening? (Select all that apply.)
Hard
- AIt can enable people to make lifestyle choices to reduce the chance of disease developing.
- BIt enables potential parents to choose whether or not to have biological children.
- CIt enables people to participate in research and clinical trials.
- DIt guarantees that a person will not develop a genetic disorder.
- EIt always lowers the cost of life insurance.
34.Which of the following are potential disadvantages of genetic screening? (Select all that apply.)
Hard
- AThere may be nothing positive that can be done in response to the results.
- BThe results may lead to higher life insurance prices.
- CIt may lead to genetic discrimination.
- DParents may be pressured into not having children.
- EIt always provides a cure for the condition screened for.
35.Why is autoradiography used in DNA probe testing?
Medium
- ATo detect the position of radioactive labels on the nylon membrane.
- BTo make the DNA fragments single-stranded.
- CTo amplify the DNA using PCR.
- DTo cut the DNA into fragments.
36.DNA probes are usually attached to a radioactive or fluorescent label.
Easy
True or false?
37.Which of the following are correct about variable number tandem repeats (VNTRs)? (Select all that apply.)
Hard
- AThey are short sequences of repeated bases.
- BThey are found within non-coding sections of DNA.
- CThey are useful in genetic fingerprinting because the number of repeats varies between individuals.
- DThey code for proteins.
- EThey are the same length in all individuals.
38.In gel electrophoresis, why do smaller DNA fragments move farther than larger fragments?
Medium
- AThey move more quickly through the tiny pores in the gel.
- BThey are more negatively charged than larger fragments.
- CThey are attracted to the negative electrode.
- DThey have a higher mass-to-charge ratio.
39.Which of the following is a correct application of genetic fingerprinting?
Medium
- ADetermining paternity.
- BProducing insulin from bacteria.
- CCuring cystic fibrosis.
- DSynthesising genes in a gene machine.
40.Which of the following are concerns associated with growing genetically modified crops? (Select all that apply.)
Hard
- ADevelopment of resistance in insects and weeds.
- BPollen from GM crops may contaminate nearby non-GM crops.
- CReduction in biodiversity.
- DGM crops always require more pesticide spraying.
- EGM crops cannot be labelled.
41.Name the enzyme that catalyses the reaction that reverses transcription, using mRNA as a template to make cDNA.
Easy
42.Name the process by which two complementary single-stranded DNA molecules combine through base pairing.
Medium
43.In a standard PCR run of 20 cycles, how many DNA molecules are produced from a single starting molecule? Give your answer as a whole number.
Medium
44.Complete the sentence about the polymerase chain reaction by filling in the missing words.
Easy
During the first stage of PCR, the DNA is heated to ____ °C to break the ____ bonds between the two strands.
45.Complete the sentence about obtaining DNA fragments.
Medium
The enzyme ____ is used to synthesise complementary DNA from mRNA, and the enzyme ____ is then used to convert the single strand of cDNA into a double-stranded DNA molecule.
46.The genetic code can be described as universal. Explain the meaning of this.
Hard
47.State and explain the purpose of primers in the polymerase chain reaction.
Hard
48.Describe how reverse transcriptase enzymes can be used to produce cDNA.
Hard
49.Explain why Taq polymerase is the enzyme used in the elongation stage of the PCR cycle.
Hard
50.Describe the process of genetic screening.
Hard
51.In a standard PCR run of 20 cycles, how many DNA molecules are produced from a single starting molecule? Give your answer as a whole number.
Medium
- A943718
- B1048576
- C1153434
- D1258292

Notes de révision

## Recombinant DNA Technology - **Genetic code is universal**: almost every organism uses the same four bases A, T, C & G; the same codons code for the same amino acids in all living things. This means genetic information is transferable between species. - **Recombinant DNA (rDNA)**: altered DNA with introduced nucleotides from another source. A **transgenic organism** contains nucleotide sequences from a different species. Any organism with introduced genetic material is a **genetically modified organism (GMO)**. - Steps to genetically engineer an organism: identify DNA fragment/gene → isolate → multiply (PCR) → transfer using a **vector** (plasmids, viruses, liposomes) → identify cells with new DNA using a **marker** → clone. - **Enzymes needed**: restriction endonucleases, ligase, reverse transcriptase. **Vectors** deliver DNA. **Markers** code for identifiable substances (e.g. GFP fluoresces under UV; GUS transforms colourless substrates into coloured/fluorescent products). ## Producing Fragments of DNA - **Restriction endonucleases**: cut DNA at specific restriction sites (e.g. HindIII binds AAGCTT). They cut the sugar-phosphate backbone to give **sticky ends** (one strand longer) or **blunt ends**. Sticky ends form hydrogen bonds with complementary sequences cut by the same enzyme. - **Reverse transcriptase**: uses mRNA as a template to make single-stranded **cDNA**; DNA polymerase then converts it to double-stranded DNA. Advantage: mRNA (and cDNA) does not contain introns; specialised cells make specific mRNA. - **Gene machine**: artificial synthesis of genes using nucleotides, based on known genetic code. ## Investigating Specificity of Restriction Enzymes - **Gel electrophoresis** separates DNA by size/mass and net charge. DNA is negatively charged (phosphate groups) so moves towards the positive electrode. Smaller fragments move faster and farther. - Restriction enzymes are highly specific, so same DNA sample always produces same number/size of fragments. Unknown enzyme can be identified by comparing band patterns. ## Polymerase Chain Reaction (PCR) - **In vitro** method of DNA amplification; produces large quantities from very small samples. - Each cycle: **Denaturation** (95°C – breaks hydrogen bonds), **Annealing** (50–60°C – primers bind), **Elongation/Extension** (72°C – Taq polymerase builds complementary strands). - Requires: target DNA/RNA, primers (forward and reverse – short single-stranded DNA complementary to 3’ end), DNA polymerase (Taq – thermostable, optimum high temperature), free nucleotides, buffer solution (optimum pH). - 20 cycles → million copies. ## Culture of Transformed Host Cells - **In vivo** gene cloning using bacteria (rapid multiplication, easy to culture). - Steps: isolate DNA fragment (add promoter/terminator) → insert into vector using restriction endonucleases and ligase → transport into bacterial host cells (transformed host cells) → bacteria multiply → identify transformed bacteria using marker genes → culture remaining bacteria; gene is cloned each division. - **Recombinant proteins** (e.g. insulin) produced using this method. ## Uses of Recombinant DNA Technology - **Recombinant proteins**: produced using microorganisms (bacteria, yeast, animal cells). Eukaryotic cells preferred for post-translational modification. - **Insulin**: human insulin gene inserted into bacterial plasmids; used to treat diabetes. Advantages: identical to human insulin, reliable supply, fewer ethical concerns, fewer rejection problems, cheaper. - **Factor VIII**: produced by genetically modified hamster kidney/ovary cells; treats haemophilia. Advantages: fewer ethical concerns, less risk of infection, greater production rate. - **Gene therapy**: alters genetic material to treat/cure diseases. Somatic gene therapy targets body cells (not inherited). **Ex vivo** (cells modified outside body) and **in vivo** (vector inside body). Germline gene therapy illegal in humans. - **SCID**: treated with ex vivo somatic gene therapy using virus to transfer normal ADA allele into T-lymphocytes. - **Agriculture**: GM crops resistant to herbicides/pests, enriched in vitamins. Example: Bt toxin in cotton. Benefits over selective breeding: faster, all organisms have desired characteristic, can come from different species. Ethical/social concerns: long-term health effects, labelling, contamination, biodiversity, cost to farmers. ## DNA Probes & DNA Hybridisation - **DNA probe**: short single-stranded DNA with known base sequence complementary to specific allele; attached to radioactive or fluorescent label. - **DNA hybridisation**: two complementary single-stranded DNA molecules combine through base pairing to form double-stranded DNA. - Process: cell sample → extract/purify DNA → amplify by PCR → digest with restriction endonucleases → separate by gel electrophoresis → transfer to nylon membrane → make single-stranded → add labelled probes → wash → detect (UV for fluorescent, autoradiography for radioactive). - If label shows, harmful allele present; if not, absent. Test only for one specific allele. ## Screening Patients - **Genetic screening**: testing embryo, fetus or adult to analyse DNA. Samples from tissue, chorionic villus sampling, amniocentesis. - Example: BRCA1/BRCA2 – faulty alleles increase risk of breast/ovarian cancer. Advantages: preventative measures, earlier screening, research participation. - Advantages: lifestyle choices, reproductive decisions, research. Disadvantages: no positive action, insurance issues, genetic discrimination, pressure, designer babies, religious objections. ## Genetic Counselling & Personalised Medicine - **Genetic counselling**: helps individuals understand results; discusses chances of inherited disease, lifestyle changes, treatments, chance of child with disease, termination, financial implications, ethical issues. - **Personalised medicine**: targeted drugs and synthetic tissues; uses genomic medicine (information about individual's genes to influence clinical care). ## Variable Number Tandem Repeats (VNTRs) - Short repeated base sequences in non-coding DNA; useful for genetic fingerprinting because they vary in number between individuals. ## Gel Electrophoresis - DNA fragments placed in wells near negative electrode; electric current applied; DNA moves towards positive electrode; smaller fragments travel faster/farther; bands visualised by staining, radioactive markers or fluorescent probes. ## Genetic Fingerprinting - Uses VNTRs; DNA cut into fragments, separated by gel electrophoresis, transferred to membrane, probed. Applications: crime scene investigations, paternity testing, identifying relationships. ## Key Terms - **Recombinant DNA (rDNA)**: DNA altered by introducing nucleotides from another source. - **Transgenic organism**: contains nucleotide sequences from a different species. - **GMO**: any organism with introduced genetic material. - **Restriction endonucleases**: enzymes that cut DNA at specific recognition sites. - **Sticky ends**: uneven cuts producing single-stranded overhangs that can form hydrogen bonds with complementary sequences. - **Reverse transcriptase**: enzyme that synthesises cDNA from mRNA. - **cDNA**: complementary DNA made from mRNA. - **Vector**: vehicle (plasmid, virus, liposome) used to deliver DNA into a cell. - **Marker gene**: gene coding for identifiable substance (e.g. GFP, GUS) used to identify transformed cells. - **PCR**: in vitro method of DNA amplification. - **Taq polymerase**: thermostable DNA polymerase used in PCR. - **Primer**: short single-stranded DNA complementary to 3’ end of target DNA; defines region to be amplified. - **DNA probe**: short single-stranded DNA with known base sequence complementary to specific allele; labelled. - **DNA hybridisation**: complementary single-stranded DNA molecules combine through base pairing. - **Genetic screening**: testing DNA of embryo, fetus or adult to analyse for specific alleles. - **Genetic counselling**: advice and information given to individuals about genetic test results and implications. - **Personalised medicine**: targeted drugs and treatments based on individual's genetic information. - **VNTRs**: variable number tandem repeats; short repeated sequences in non-coding DNA used in genetic fingerprinting. - **Gel electrophoresis**: technique separating DNA fragments by size and charge using an electric current. - **Gene therapy**: altering a person's genetic material to treat or cure disease. - **Ex vivo gene therapy**: cells modified outside the body then returned. - **In vivo gene therapy**: vector delivers gene directly inside the body. - **Somatic gene therapy**: targets body cells; not inherited. - **Germline gene therapy**: targets gametes; illegal in humans. - **Recombinant proteins**: proteins produced by genetically modified organisms. - **Transformed host cells**: cells that have taken up recombinant DNA.

Recombinant DNA (rDNA): DNA that has been altered by introducing nucleotides from another source.
Transgenic organism: An organism that contains nucleotide sequences from a different species.
Genetically modified organism (GMO): Any organism that has introduced genetic material.
Restriction endonucleases: Enzymes that cut DNA at specific recognition sites, producing sticky or blunt ends.
Sticky ends: Uneven cuts in DNA that result in one strand being longer than the other, allowing hydrogen bonds to form with complementary sequences.
Reverse transcriptase: Enzyme that synthesises a single strand of complementary DNA (cDNA) from mRNA.
cDNA: Complementary DNA made from mRNA using reverse transcriptase; does not contain introns.
Vector: A vehicle (e.g. plasmid, virus, liposome) used to deliver DNA fragments into a cell.
Marker gene: A gene that codes for an identifiable substance (e.g. GFP, GUS) used to identify transformed cells.
Polymerase chain reaction (PCR): An in vitro method of DNA amplification used to produce large quantities of specific DNA or RNA fragments.
Taq polymerase: A thermostable DNA polymerase used in PCR; does not denature at high temperatures.
Primer: A short sequence of single-stranded DNA complementary to the 3’ end of the DNA or RNA being copied; defines the region to be amplified.
DNA probe: A short length of single-stranded DNA with a known base sequence complementary to a specific allele; attached to a radioactive or fluorescent label.
DNA hybridisation: The process where two complementary single-stranded DNA molecules combine through base pairing to form a double-stranded DNA molecule.
Genetic screening: The testing of an embryo, fetus or adult to analyse their DNA for specific alleles.
Genetic counselling: Advice and information given to individuals to help them understand and process genetic test results.
Personalised medicine: The development of targeted drugs and treatments based on an individual's genetic information.
Variable number tandem repeats (VNTRs): Short sequences of repeated bases found within non-coding sections of DNA; useful for genetic fingerprinting.
Gel electrophoresis: A technique that uses an electric current to separate DNA fragments according to their size/mass and net charge.
Gene therapy: Using mechanisms to alter a person's genetic material to treat or cure diseases.
Ex vivo gene therapy: The new gene is inserted via a virus vector into cells outside the body, which are then returned to the patient.
In vivo gene therapy: The new gene is inserted via a vector into cells inside the body.
Somatic gene therapy: Gene therapy that targets body cells; changes are not inherited by future generations.
Germline gene therapy: Gene therapy that targets gametes; illegal in humans as changes could be inherited.
Recombinant proteins: Proteins produced by genetically modified organisms, e.g. insulin, Factor VIII.
Transformed host cells: Cells that have taken up recombinant DNA, e.g. bacteria containing modified plasmids.

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