3.5 ~ Genetic modification and biotechnology

DNA profiling

• Involves comparison of DNA

• PCR and gel electrophoresis can be used to build DNA profiles

• DNA profiling is a technoique by which individuals can be identified and compared via their respective DNA profiles

• This can create a unique ‘fingerprint’ of an individual that can be used in forensic science to help solve legal and criminal cases such as testing for paternity, genealogy, identifying suspects from crime scene etc.

Comparison of DNA

• Amplitification of DNA sample using PCR

• Cutting DNA at various loci to produce DNA fragments

• Separation of DNA fragments using gel electrophoresis

• The more marker that are used to build a DNA profile, the more reliable a comparison will be

• Seperation of fragment creates a pattern of bands that ar unique to each individual

  • These can then be compared with other samples

Use of PCR

• Make a large number of copies of a DNA sample

• Only a tiny amount of DNA is needed

  • This technique is very useful for amplifying of very small samples of DNA

• PCR is used to copy specific sequences in the DNA

  • Primers are selected based on the sequences that scientists want to amplify

Gel Electrophoresis

• A technique used to separate molecules by size and charge

• Uses an arose gel that contains pores that are just big enough for fragments of DNA to move through

• The gel has wells at one end where the DNA samples are loaded

• Gel is placed in a tank with an electrolyte solution

  • A voltage is placed across the tank so that the negative electrode is at the end of the gel where the wells are placed and the positive electrode is at the other end

• When the voltage is applied, the negatively charged DNA molecules will move through the gel towards the positive electrode

Separating fragments

• The smaller the molecules of DNA, the further they will travel through the gel

  • Longer molecules will face more resistance and will not move as far

• A DNA ladder can be run in one of the lanes

  • This obtains fragments of known lengths that can be used to estimate the size of the fragments in the gel

Process of gel electrophoresis

• DNA is cut into fragments by enzymes called restriction endoncucleases

  • Different restriction enzyme cut at different points in the DNA

• Samples are added to wells in the gel and an electric field applied across the gel

  • DNA fragments will move towards the positive electrodes

  • DNA fragments are negatively charged

Describe the application of DNA profiling to determine paternity:

1. DNA from child, mother and possible fathers used to establish paternity

2. For legal reasons e.g. divorce/ inheritance

3. For personal reasonns

4. DNA copied using PCR

5. DNA cut using restriction enzymes

6. Gel electrophoresis is used to separate DNA fragments

7. Pattern of band is produced in gel

8. Analysed for matches between child with mother and possible father

9. Half the child’s bands will match the father

Outline the process of DNA profiling:

1. Sample of DNA obtained from person

2. PCR used to amplify copies of DNA

3. Using TAQ polymerase

4. Tandem repeats used

5. Gel electrophoresis used to separate DNA

6. Separation according to length of fragments

7. Pattern of bands is unique to the individual

8. Used in crime investigation/ paternity test

Denaturation

DNA sample is heated to 95 which break the H bonding between the strands = breaking

Annealing

Sample is cooled to 54 and mixed with DNA primers which attach to opposite ends of the target sequence by complementary base pairing- essential to initiate replication as optimum temp for DNA strands to reanneal to each other

Extension

At 72 Taq DNA polymerase adds free DNA nucleotides to the strands of the DNA, using the primers as a starting point

Genetic modification

• Milk containing spider silk protein is produced by goats

  • Spider silk is immensely strong

• Carried out by gene transfer between species

• Genoese from snapdragons have been introduced into tomato plants to generate purple fruits

• Human insulin produced by bacteria for diabetics

• Golden rice is colour yellow as it contains B-carotene

  • A precursor of vitamin A

Gene transfer

• Genetic code is universal

• Number of chromosomes varies but DNA code is the same

• Plasmid is taken out of bacterial cell

• Gene in eukaryotic cell is isolated

• Restriction enzymes used to cut plasmid and specific gene - creating complimentary sticky ends

• Enzyme DNA ligase

• Plasmid and DNA cut at same nuclear points

Use of plasmids

• Some restriction endonucleases have the very useful property of creating ‘sticky ends’ when they cut DNA

• This mantis they leave short single-stranded ends overhanging when they cut the DNA

  • The sticky ends on either sides of the cut fragment will be complimentary

• If the different pieces of DNA are cut suing the same restriction enzyme, the sticky ends will be the same and therefore, complimentary

• This makes it very easy to lígate DNA fragments back together as the ends of each fragment will be complementary

Explain the process of genetically modifying bacteria:

1. Genetic modification carried out by gene transfer between species

2. Genes transferred from one organism to another produce the same protein/amino acid sequence

3. Due to university of genetic code

4. mRNA for required gene extracted

5. DNA copies of mRNA made using reverse transcriptase

6. PCR used (to amplify DNA to be transferred)

7. Genes transferred from one species to another using a vector

8. Plasmid acts as a vector to transfer genes to bacteria

Bt crops:

• Insect pests that feed on crops reduce the yield of crops for farmers

  • Insecticides are chemicals that kill insects; however can be harmful to the environment and humans

• Variteties of crops have been genetically modified to produce a toxin that kill insects - the Bt toxin from the bacterium Bacillus thuringiensis

  • These crops mean that less/ no insecticide needs to be used on crops

• Many varieties of Bt crops have been produced including corn and maize

• GM crops that produce the Bt toxin produce the toxin in all parts of the plant, including the pollen

Risk on monarch butterflies

• Concerns have been raised about the effect of Bt toxin on non-target insect species, including the monarch butterfly

• The larvae of the monarch butterfly feed on plants that sometimes grow close to the Bt crops

  • Some of the pollen from Bt crops is blown onto the plants that the monarch butterfly feed on

  • This pollen is toxic to the monarch butterfly, killing them