StemUp: AQA A level Biology 3.8.4 Gene technologies allow the study and alteration of gene function allowing a better understanding of organism function and the design of new industrial and medical processes

Describe the basics of recombinant DNA technology (3)

- The transfer of DNA fragments from one organism or species to another

- The transferred DNA can be transcribed / translated into proteins

- Within the cells of the recipient (transgenic) since the genetic code is universal

How can fragments of DNA be produced by the conversion of mRNA to complementary DNA (cDNA)? (4)

1. mRNA is isolated from a cell that readily synthesises the protein coded for by the desired gene

2. Mix the RNA with DNA nucleotides and reverse transcriptase

3. The reverse transcriptase will use the mRNA as a template to synthesise a single strand of cDNA

4. DNA polymerase forms a second strand of DNA using cDNA as a template

How can fragments of DNA be produced by using restriction enzymes to cut a fragment containing the desired gene from DNA? (3)

1. Different restriction endonucleases cut DNA at specific sequences of bases called a 'recognition sequence'

2. The shape of the recognition site is complementary to the active site of the endonucleases

3. Some restriction enzymes cut in a staggered fashion and 'sticky ends' are formed

How can fragments of DNA be produced by using a gene machine? (2)

1. Gene machine will synthesise fragments of DNA from scratch without the need for a pre-existing DNA ‎template

2. DNA fragments are therefore produced quickly and accurately without ‎‎introns

What is the role of promoter and terminator regions in the in-vivo method to amplify DNA fragments? (2)

- Promotor regions will tell the RNA polymerase when to start transcription to produce mRNA

- Terminator regions tell the RNA polymerase when to stop

What is the role of restriction endonuclease in the in-vivo method to amplify DNA fragments? (3)

- Restriction endonucleases will transfer DNA fragments from one organism or species, to another

- This DNA can then be transcribed / translated into proteins within cells of the recipient (transgenic) organism

- Since the genetic code is universal

Describe how the in-vivo method of amplifying DNA fragments is carried out (6)

1. Promoter and terminator regions are added to the DNA fragment to ensure proper transcription within the host cell

2. Both the vector DNA and the DNA fragment are cut using the same restriction endonuclease, producing complementary sticky ends

3. The DNA fragment is then inserted into the vector DNA. The complementary sticky ends of the DNA fragment and vector align

4. DNA ligase is used to join the DNA fragment to the vector DNA by forming phosphodiester bonds between adjacent nucleotides, creating a recombinant DNA molecule.

5. The recombinant vector is introduced into a host cell, which takes up the transformed vector containing the DNA fragment of interest.

6. Marker genes may also be inserted into the vector alongside the target gene to identify which host cells have successfully taken up the vector with the desired gene

What does the reaction mixture used in PCR contain? (4)

- DNA fragments

- DNA polymerase

- DNA primers

- Free DNA nucleotides

Describe how the in-vitro (PCR) method of amplifying DNA fragments is carried out (5)

1. Reaction mixture is heated to 95 degrees to separate DNA fragments by breaking the hydrogen bonds between bases

2. Reaction mixture is cooled to 55 degrees to allow primers to bind to the DNA fragment template strand by forming hydrogen bonds between complementary bases

3. Mixture is heated back to 72 degrees as this is the optimum temperature for DNA polymerase

4. DNA polymerase joins adjacent complementary nucleotides, by forming phosphodiester bonds

5. Cycle is repeated until we run out of nucleotides / primers

What are some examples of recombinant DNA technology in agriculture? (3)

- GM crops that are resistant to herbicides

- GM crops that are resistant to insect attack

- GM crops with added nutritional value e.g. golden rice

What are some examples of recombinant DNA technology in medicine? (3)

- GM bacteria that produce recombinant human proteins e.g. insulin, which is more ethically acceptable than insulin formed by pancreas from pigs

- GM animals / plants that produce pharmaceuticals, which are cheaper to produce

- Gene therapy, which uses new DNA which contains healthy / functional alleles to overcome the effect of faulty alleles e.g. for cystic fibrosis

What are the benefits of recombinant DNA technology? (4)

- GM crops increase yield

- This increases global food production which combats risk of malnutrition

- Gene therapy has the potential to cure many genetic disorders

- Production of pharmaceutical drugs is cheaper

What are the drawbacks of recombinant DNA technology? (4)

- GM crops can have an effect on food webs

- Which reduces biodiversity

- Gene therapy's long term effect are not known

- Side effects could for example, cause cancer

How is DNA prepared in the genetic screening process? (2)

1. DNA is extracted using restriction enzymes that cut the DNA at specific base sequences

2. The DNA is then amplified by PCR

How are DNA fragments separated during genetic screening? (2)

- Separated by gel electrophoresis

- According to their length

What happens to the DNA fragments after gel electrophoresis in genetic screening? (2)

1. The DNA fragments are transferred to a nylon membrane

2. They are treated to form single strands with exposed bases

What is the role of a labelled DNA probe in genetic screening? (1)

It will hybridize with its target allele on the nylon membrane

How is the presence of the bound DNA probe detected in genetic screening? (2)

By exposing the membrane:

- To UV light (if fluorescently labelled probes are used)

- by using autoradiography (if radioactive probes are used)

What are some examples for the uses of genetic screening? (3)

- Screening patients for heritable condition e.g. finding out if they are a carrier to a disease such as cystic fibrosis

- Screening patients for drug responses i.e. how their body will respond to certain drugs

- Screening patients for health risks e.g. for alleles that may indicate a risk to high blood cholesterol levels / chance of breast cancer

What are the roles of a genetic counsellor? (5)

- Explain the results of screening / consequences of a disease

- Discuss treatments available for genetic conditions

- Discusses lifestyle choices / precautions that might reduce the risk of a genetic condition developing (eg. regular screening for tumours)

- Explain probability of condition/alleles being passed onto offspring

- Which would enable patients to make an informed decision about having children

Describe personalised medicine (3)

- Medicine that is tailored to an individual's genotype / DNA with the use of genetic screening

- This increases the effectiveness of treatment eg. identifying a particular mutation / allele causing cancer

- Which will allow you to treat the patient with specific drugs you know will be effective

What are some arguments for and against the use of genetic screening? (5)

What are VNTRs? (2)

- Variable number tandem repeats

- Repeating sequences of nucleotides / bases found within non-coding sections of DNA at many sites throughout the genome

What do VNTRs allow for? (2)

- Allow for the production of a virtually unique genetic fingerprint

- As the probability of two individuals having the same VNTRs is very low

How is DNA prepared for genetic fingerprinting in the analysis of VNTRs? (2)

- DNA is extracted and amplified by PCR.

- Restriction enzymes then cut the DNA at specific base sequences near VNTRs

How are DNA fragments containing VNTRs separated during genetic fingerprinting? (2)

- Separated by gel electrophoresis according to their length

- With shorter VNTRs traveling further

What happens to the DNA fragments after gel electrophoresis in genetic fingerprinting? (2)

- DNA fragments are transferred to a nylon membrane

- Treated to form single strands with exposed bases

What is the role of a labelled DNA probe in genetic fingerprinting? (1)

It will hybridize with its target VNTRs on the nylon membrane

Describe the principle method of gel electrophoresis (4)

1. DNA samples are loaded onto wells porous gel and are covered in a buffer solution which conducts electricity

2. An electrical current is passed through the sample

3. DNA is negative charged so it will moves towards the positive electrode

4. Shorter DNA fragments travel faster so travel further

What are the uses of genetic fingerprinting in identifying genetic relationships? (2)

1. More closely related = more similar VNTRs = more similarities in genetic fingerprints

2. Paternity testing - father should share around half of VNTRs/bands and ones that don't come from mother must be from father

What are the uses of genetic fingerprinting in identifying genetic variability within a population? (2)

- Differences in VNTRs arise from mutations

- The more differences there are, the greater the diversity ‎within a population

What are the uses of genetic fingerprinting in forensic science? (2)

1. They compare the genetic fingerprints of suspects to DNA at crime scenes

2. If many bands match, a person is linked to a crime scene / likely to be present

What are the uses of genetic fingerprinting in medical diagnosis? (2)

- Useful when specific mutation not known or several could have caused disorder

- Identifies a broader altered genetic pattern, ie a risk factor

What are the uses of genetic fingerprinting in animal and plant breeding? (2)

- Shows how closely related two individuals are, so that inbreeding can be avoided

- Select pairs with dissimilar genetic fingerprints

How could you estimate the size of DNA fragments with gel electrophoresis (2)

- Carry out gel electrophoresis on DNA fragments of known lengths

- Compare the length to position of unknown fragments