History of DNA profiling Flashcards
Properties of good genetic marker for forensic purposes
- Highly polymorphic (tell between individuals)
- Cheap and easy to characterise
- Profiles simple to interpret and compare
- Low mutation rate
- Ability to automate
- Deal well with mixed samples
Blood Groups
- First genetic tool for distinguishing between individuals
- Not very informative- poor power of discrimination
- Generally, need to combine systems such as ABO, RH etc
- Quick analysis- a few mins
- Need large samples esp if combining techniques
- Blood groups are manifestation of differences on DNA but limited because need to be function molecules
ABO blood groups
- Antibody- protein produced by immune system to recognise and neutralise pathogens
- Antigen- target for antibodies- foreign substance that provokes immune response
- ABO system based on antigenic substances on the surface of RBCs
- ABO antigens are sugars
- Types are, A B AB and O
- Groups can be incompatible for transfusion
- O universal donor
Another system of DNA profiling
rhesus System and polymorphic protein systems were utilised in forensics as well
Back ground to DNA profiling
- 60s and 70s paved the way for DNA fingerprinting
- Restriction enzymes
- Electrophoresis
- Southern blotting
- Used to detect DNA polymorphisms
restriction enzymes
- Enzymes from bacteria
- Cut dna at a restriction site
- Essential for genic manipulations
- Restriction sites are usually dna sequences of 4-6 nucleotides that the enzyme scans the genome for
What is Gel electrophoresis
Separation of DNA fragments by restriction enzymes
Gel electrophoresis
1- DNA samples containing fragments of different sizes are placed in agarose gel
2- an electrical current is passed through the gel
3- all DNA fragments move toward the positive pole, with smaller fragments migrating faster
4- After electrophoresis, fragments of different sizes have migrated different distances
5- a dye specific for nucleic acids is added to the gel
6- under UV light the DNA fragments will glow according to the dye colour
what is Southern blotting
DNA fragment detection
southern blotting
1- DNA is cleaved by restriction enzymes and transferred to an agarose gel. The fragments are separated by gel electrophoresis.
2- the gel is soaked in an alkali solution to denature the double stranded DNA
3- a membrane is positioned on top of the gel
4- weight is placed on top of the membrane
5- DNA is carried onto the membrane as the buffer is drawn up through the gel
6- DNA on the membrane is fixed
7- the membrane is placed in a hybridisation bottle with a solution that contains a radioactively labeled probe, and gently rotated
8- the probe binds to complementary DNA fragments on the membrane
9- autoradiography detects fragments with the probe attatched
Restriction fragment length polymorphisms
- hereditary
- Usually determined by presence/absence of restriction sites
- The substation of a single base in the dna sequence can either create or destroy a restriction site
- The creation of a new restriction site will create 2 smaller fragments where before there was 1 larger fragment
- Destruction of a restriction site would have the opposite effect
- Varation = RFLP
Variable number tandem repeats
- Also called minisatellites
- a type of RFLP
- Regions of dna that contain dna sequences that are repeated
- Number of repeats varies from person to person
Multi locus probes
- Probes that recognise sequences belonging to length polymorphism at a number of different genetic loci simultaneously
- Produces a complex pattern of bans called a ‘dna fingerprint’
single locus probes
- Probes that are specific for only one minisatellite
- Need smaller amount of dna
- Generate simpler results
- Increase power of discrimination by combining various SLPs because its at a single location
Limitations of RFLPs
- Time consuming
- A lot of expertise needed to produce result and interpret
- Large amount of starting material needed
- Good quality DNA needed (not degraded)
