DNA PROFILING AND PCR – TOPIC 6
1. DNA PROFILING (fingerprinting)
- Used to study DNA
- Obtain tissue/cell sample from source ‘X’ – e.g. blood, saliva, samen
- Cells are broken down in buffer solution
- Protease enzymes used to remove proteins and ethanol is added to precipitate out the DNA
- DNA profiling assumes that every individual is unique – apart from identical twins
- It analyses the introns and non-coding DNA (very reliable)
- There is a large number of introns and non-coding blocks, so many combinations of
genes/DNA/alleles at locus.
2. PCR – Polymerase chain reaction
- Multiple copies of DNA are made
- Denature the DNA – separate the DNA double helix into 2 separate strands – 90-95
- DNA primers and nucleotides are needed
- Primers attach to the 2 separate strands (1 on each strand) – 50-65
- 2 polymerase molecules attach to the 2 primers on the 2 separate strands and move along
the strand – 70-75
- This cycle needs to be repeated several times to make several multiple copies of DNA –
amount large enough to be examined
3. Creating DNA fragments
- Restrictions enzymes are used to break the DNA
- They cut the DNA at specific places of sequences known as restriction sites
- To produce DNA fragments
4. Separation of DNA fragments
- Load the DNA fragments onto agarose gel plate
- Electric current is applied across the gel plate – potential difference is used
- DNA (negatively charged) moves towards the anode/positive terminal
5. Southern blotting
- Transfer the profile to a more durable material such as nylon membrane
- The samples will be drawn by the flow of water diffusing into a drier material into the nylon
sheet
- The DNA fragments show up as bands
- DNA bands can be visualised by using gene probes or fluorescent staining, which can be seen
with UV light
,6. How are 2 DNA profiles compared?
- Compare the total number of bands
- Position of bands
- Size/width of bands
- Common bands contain similar DNA
7. Similarities in DNA of ‘A’ and ‘B’ using PCR. What do these similarities suggest?
- Same number of chromosomes
- Number of bands that match indicate similarities in DNA
- Common bands contain similar DNA
- Similar DNA indicates closeness of relationship because genes are sections of DNA and genes
are codes for proteins
- More similar patterns à more likely to have a common ancestor
8. DNA polymerase from human sources is not suitable for use in PCR. Why?
- Human enzymes will not work at high temp, which is above 37 – denaturation of proteins
9. Plants cannot be identified using PCR and DNA profiling. Why?
- Xylem is made from dead material/tissue – no DNA – no nucleus
10. Suggest why DNA profiles may not be conclusive
- DNA profiling has several stages
- Contamination can arise at any stage
- Only a small portion of the DNA is analysed
- There might be a possibility of 2 identical profiles from unrelated individuals
11. Suggest how DNA profiling could be useful to scientists who examine fossils of animals and
plants
- Comparison of the DNA profiles/made between DNA from fossils and other organism
(number/width/size of bands) used to find genetic relationship between the two organisms
and DNA similarities to show how closely related they are
- DNA profiling could be used in taxonomy to understand evolutionary lines and to determine
common ancestor
, DETERMING TIME OF DEATH
1. BODY TEMPERATURE
- Average body temperature is between 36.2 and 37.6
- Temperature of body drops after death – linked to time after death
- Chemical reactions that produce heat stop causing the body to cool
- Factors, which affect temperature are: environmental temp, body size, clothing
- It is useful because time of death can be calculated if the ambient temperature is known
- Ambient temp – average air temp around body
- Only useful for a short period of time following death – 24 hours
- This is because, the extent of temp depends on ambient temp
- Ambient temp fluctuates over time – does not stay constant
- Sooner after death that the temp is taken, the more accurate the estimate of time of death
- Calculations of time of death based on the average body temp – 37
- This also depends on the time of the day
2. RIGOR MORTIS
- The oxygen dependent reactions in the body will stop
- There is not enough oxygen in the body that is required for aerobic respiration
- So respiration in the cells become anaerobic – produces lactic acid
- No ATP produced needed for muscle contraction
- Muscles cannot contract – they become fixed/stiff
- The environment becomes too acidic for enzymes – enzyme activity decreases – PH drops
3. FORENSIC ENTOMOLOGY
- Used to determine time of death
- Insects occur in the succession of the decomposing body
- Forensic entomologists record the type of insect found on the body (fly, wasp)
- Identify the species of the insect and the stage of lifecycle that they are in
- External factor that affects forensic entomology is temperature
- This can be compared to current data to estimate time of death
4. DECOMPOSITION
- Micro-organisms such as bacteria act as decomposers
- Environmental temperature increases the rate of decomposition
- This is because temp increases the kinetic energy of molecules – increases the number of
collisions between enzymes and substrates – more enzyme-substrates formed – increases
enzyme activity
- Also increases the rate at which bacteria grow (bacteria contains enzymes)
- So, tissues begin to denature due to action of enzymes and bacteria
- Above a certain temp rate (optimum temp), decomposition decreases
- Enzymes denature and bacteria are killed
1. DNA PROFILING (fingerprinting)
- Used to study DNA
- Obtain tissue/cell sample from source ‘X’ – e.g. blood, saliva, samen
- Cells are broken down in buffer solution
- Protease enzymes used to remove proteins and ethanol is added to precipitate out the DNA
- DNA profiling assumes that every individual is unique – apart from identical twins
- It analyses the introns and non-coding DNA (very reliable)
- There is a large number of introns and non-coding blocks, so many combinations of
genes/DNA/alleles at locus.
2. PCR – Polymerase chain reaction
- Multiple copies of DNA are made
- Denature the DNA – separate the DNA double helix into 2 separate strands – 90-95
- DNA primers and nucleotides are needed
- Primers attach to the 2 separate strands (1 on each strand) – 50-65
- 2 polymerase molecules attach to the 2 primers on the 2 separate strands and move along
the strand – 70-75
- This cycle needs to be repeated several times to make several multiple copies of DNA –
amount large enough to be examined
3. Creating DNA fragments
- Restrictions enzymes are used to break the DNA
- They cut the DNA at specific places of sequences known as restriction sites
- To produce DNA fragments
4. Separation of DNA fragments
- Load the DNA fragments onto agarose gel plate
- Electric current is applied across the gel plate – potential difference is used
- DNA (negatively charged) moves towards the anode/positive terminal
5. Southern blotting
- Transfer the profile to a more durable material such as nylon membrane
- The samples will be drawn by the flow of water diffusing into a drier material into the nylon
sheet
- The DNA fragments show up as bands
- DNA bands can be visualised by using gene probes or fluorescent staining, which can be seen
with UV light
,6. How are 2 DNA profiles compared?
- Compare the total number of bands
- Position of bands
- Size/width of bands
- Common bands contain similar DNA
7. Similarities in DNA of ‘A’ and ‘B’ using PCR. What do these similarities suggest?
- Same number of chromosomes
- Number of bands that match indicate similarities in DNA
- Common bands contain similar DNA
- Similar DNA indicates closeness of relationship because genes are sections of DNA and genes
are codes for proteins
- More similar patterns à more likely to have a common ancestor
8. DNA polymerase from human sources is not suitable for use in PCR. Why?
- Human enzymes will not work at high temp, which is above 37 – denaturation of proteins
9. Plants cannot be identified using PCR and DNA profiling. Why?
- Xylem is made from dead material/tissue – no DNA – no nucleus
10. Suggest why DNA profiles may not be conclusive
- DNA profiling has several stages
- Contamination can arise at any stage
- Only a small portion of the DNA is analysed
- There might be a possibility of 2 identical profiles from unrelated individuals
11. Suggest how DNA profiling could be useful to scientists who examine fossils of animals and
plants
- Comparison of the DNA profiles/made between DNA from fossils and other organism
(number/width/size of bands) used to find genetic relationship between the two organisms
and DNA similarities to show how closely related they are
- DNA profiling could be used in taxonomy to understand evolutionary lines and to determine
common ancestor
, DETERMING TIME OF DEATH
1. BODY TEMPERATURE
- Average body temperature is between 36.2 and 37.6
- Temperature of body drops after death – linked to time after death
- Chemical reactions that produce heat stop causing the body to cool
- Factors, which affect temperature are: environmental temp, body size, clothing
- It is useful because time of death can be calculated if the ambient temperature is known
- Ambient temp – average air temp around body
- Only useful for a short period of time following death – 24 hours
- This is because, the extent of temp depends on ambient temp
- Ambient temp fluctuates over time – does not stay constant
- Sooner after death that the temp is taken, the more accurate the estimate of time of death
- Calculations of time of death based on the average body temp – 37
- This also depends on the time of the day
2. RIGOR MORTIS
- The oxygen dependent reactions in the body will stop
- There is not enough oxygen in the body that is required for aerobic respiration
- So respiration in the cells become anaerobic – produces lactic acid
- No ATP produced needed for muscle contraction
- Muscles cannot contract – they become fixed/stiff
- The environment becomes too acidic for enzymes – enzyme activity decreases – PH drops
3. FORENSIC ENTOMOLOGY
- Used to determine time of death
- Insects occur in the succession of the decomposing body
- Forensic entomologists record the type of insect found on the body (fly, wasp)
- Identify the species of the insect and the stage of lifecycle that they are in
- External factor that affects forensic entomology is temperature
- This can be compared to current data to estimate time of death
4. DECOMPOSITION
- Micro-organisms such as bacteria act as decomposers
- Environmental temperature increases the rate of decomposition
- This is because temp increases the kinetic energy of molecules – increases the number of
collisions between enzymes and substrates – more enzyme-substrates formed – increases
enzyme activity
- Also increases the rate at which bacteria grow (bacteria contains enzymes)
- So, tissues begin to denature due to action of enzymes and bacteria
- Above a certain temp rate (optimum temp), decomposition decreases
- Enzymes denature and bacteria are killed
