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)
STRs
- Microsatellites
- Carriable, repeated dna
- Analysis based on PCR
High power of discrimination
Restricted fragment length polymorphism
Multi locus probes
single locus probes
Multiplex STRs
Low power of discrimination
mtDNA
D1s80
single STR
ABO blood groups
Slow speed of analysis
Restricted fragment length polymorphism
Multi locus probes
single locus probes
mtDNA
Fast speed of analysis
Multiplex STRs
D1S80
single str
ABO blood groups
