21.1 DNA profiling
5/12/23
Genome = all the genes possessed by an individual or an organisms (ie.The
whole base sequence (e.g. includes DNA in nucleus and mitochondria)
- Approx 99% of genome same across all human beings
2 subsections of genome:
1) Exons - (2%) codes for proteins
2) Introns (98%) - may be involved in gene expression control
Dna profiling = looking for differences in individual genome
Looking at differences within Introns
- Contain satellite DNA = short repeated DNA sequences
- 2 types of satellite DNA ( Mini satellite - 20-50 base pairs long and
repeated 50 - 100 times + Microsatellite - 2-4 base pairs long and
repeated 5-15 times)
- Both types of satellite DNA work in the same way - they always appear in the
same area on chromosomes
- When we cut DNA apart we will generate a different satellite pattern
- Different people have different number of repeats → different satellite
patterns
- Dna profiling helps identify an individual & determine familial
relationships (can be used in forensics e.g. see which suspect matched
with dna from crime scene - looking at satellite pattern/ OR paternal testing)
Process of DNA profiling
1) Extract DNA from pool of blood from crime scene
2) Sometimes the pool of blood is not enough for dna. So we do PCR to
amplify DNA ( this makes lots and lots of DNA)
3) We also add some enzymes into it (which is restriction endonucleases -
these recognise the specific dna bases known as restriction sites (these
are within introns but not on satellite dna) . They will then cut the dna up
at these restriction points.
4) We then need to find a way to separate these small fragments from each
other. So we do gel electrophoresis. We use electricity for this process.
This is where we put the DNA fragments in the wells within agar jelly. (this
should be the negative side of the electrode - this is because DNA itself is
negatively charged due to phosphate groups so it will be repelled against
negative electrodes and move towards positive electrodes. You may also
put the suspects in the other wells. Another thing is that we immerse the
, agar jelly in alkaline so double stranded dna → broken into single
stranded dna. This way they can move a lot easier to the other side.
5) To see the dna we do southern blotting. This is where we put a nylon sheet
on top of the DNA. This makes separated dna fragments to be soaked up
into the sheet. WE can study the data easier this way
6) Hybridisation - this is where we add dye to it ( can be fluorescent dye or
radioactive dye). Then it should be put under uv light if using fluorescent
dye / x ray light if using radioactive dye .
7) You can then see evidence
PCR
- This is artificial DNA replication which amplifies DNA fragments for
further study/processing
- This is used when there isn't enough dna in a substance to generate a
DNA profile e.g. a strand of hair
- PCR takes place in a thermal cycler machine. This carefully controls and
changes its temp at programmed timings to trigger the diff steps in PCR
Stages:
1) Denaturation
- This takes place at 95’c
- Molecules gain KE → increased vibration to the point where nitrogenous
bases separate from their complementary bases so much that they break
the hydrogen bonds in between → separate dna strands
2) Annealing of the primers
- Temp is decreased to 55 - 60’c
5/12/23
Genome = all the genes possessed by an individual or an organisms (ie.The
whole base sequence (e.g. includes DNA in nucleus and mitochondria)
- Approx 99% of genome same across all human beings
2 subsections of genome:
1) Exons - (2%) codes for proteins
2) Introns (98%) - may be involved in gene expression control
Dna profiling = looking for differences in individual genome
Looking at differences within Introns
- Contain satellite DNA = short repeated DNA sequences
- 2 types of satellite DNA ( Mini satellite - 20-50 base pairs long and
repeated 50 - 100 times + Microsatellite - 2-4 base pairs long and
repeated 5-15 times)
- Both types of satellite DNA work in the same way - they always appear in the
same area on chromosomes
- When we cut DNA apart we will generate a different satellite pattern
- Different people have different number of repeats → different satellite
patterns
- Dna profiling helps identify an individual & determine familial
relationships (can be used in forensics e.g. see which suspect matched
with dna from crime scene - looking at satellite pattern/ OR paternal testing)
Process of DNA profiling
1) Extract DNA from pool of blood from crime scene
2) Sometimes the pool of blood is not enough for dna. So we do PCR to
amplify DNA ( this makes lots and lots of DNA)
3) We also add some enzymes into it (which is restriction endonucleases -
these recognise the specific dna bases known as restriction sites (these
are within introns but not on satellite dna) . They will then cut the dna up
at these restriction points.
4) We then need to find a way to separate these small fragments from each
other. So we do gel electrophoresis. We use electricity for this process.
This is where we put the DNA fragments in the wells within agar jelly. (this
should be the negative side of the electrode - this is because DNA itself is
negatively charged due to phosphate groups so it will be repelled against
negative electrodes and move towards positive electrodes. You may also
put the suspects in the other wells. Another thing is that we immerse the
, agar jelly in alkaline so double stranded dna → broken into single
stranded dna. This way they can move a lot easier to the other side.
5) To see the dna we do southern blotting. This is where we put a nylon sheet
on top of the DNA. This makes separated dna fragments to be soaked up
into the sheet. WE can study the data easier this way
6) Hybridisation - this is where we add dye to it ( can be fluorescent dye or
radioactive dye). Then it should be put under uv light if using fluorescent
dye / x ray light if using radioactive dye .
7) You can then see evidence
PCR
- This is artificial DNA replication which amplifies DNA fragments for
further study/processing
- This is used when there isn't enough dna in a substance to generate a
DNA profile e.g. a strand of hair
- PCR takes place in a thermal cycler machine. This carefully controls and
changes its temp at programmed timings to trigger the diff steps in PCR
Stages:
1) Denaturation
- This takes place at 95’c
- Molecules gain KE → increased vibration to the point where nitrogenous
bases separate from their complementary bases so much that they break
the hydrogen bonds in between → separate dna strands
2) Annealing of the primers
- Temp is decreased to 55 - 60’c
