- Created by: Ali
- Created on: 02-06-13 10:03
Genetic fingerprinting overview
The genome of any organism contains many repetitive, non-coding DNA bases (introns).
The repetitive sequences contained in introns are called core sequences.
In every individual length and patterns of the core sequences is unique (except in identical twins).
The more closely related two individuals are, the more similar the core sequences will be.
The five main stages of genetic fingerprinting are:
Extraction, digestion, separation, hybridisation and development.
DNA is extracted by separating it from the rest of the cell.
As the DNA amount is small, the quantity can be increased by using the Polymerase Chain Reaction.
Restriction endonucleases cut the DNA into fragments.
The enzymes are chosen for their ability to cut close to the core sequences without altering them.
Gel electrophoresis is used to separate the fragments according to size under the influence of an electrical voltage.
The gel is immersed in alkali to separate the double nucleotide-strands of DNA.
Each single strand is transferred by southern blotting onto a nylon membrane.
Southern blotting is achieved as follows:
- Thin nylon membrane is laid over the gel
- The nylon membrane is covered with sheets of absorbent paper, which draws up the liquid containing DNA by capillary action
- This transfers DNA fragments to the nylon membrane in the same positions they occupied on the gel.
- The DNA fragments are fixed to the nylon membrane using Ultraviolet light.
Flourescent DNA probes bind with core sequences
The probes have base sequences that are complementary to core sequences and bind to the DNA under specific conditions (temp., pH and light).
Different probes bind to different core sequences
An X-ray film is now put over the nylon membrane.
The radiation from the probes allows the position of the fragments after electrophoresis to be seen.
The pattern of the bands is unique to every individual (except identical twins).
Interpreting the results
An automatic scanning machine can calculate the length of the DNA fragments. This is done using data obtained by measuring the distances travelled during electrophoresis results by known lengths of DNA.
The odds are calculated for somebody else having the same pattern: the closer the match between two patterns, the greater the chance of the DNA coming from the same person..
Uses of DNA fingerprinting
Genetic fingerprinting can be used to: -
- test for paternity. Half the DNA of an individual comes from their mother and the other half from their father, each band on a DNA fingerprint should have a corresponding band on the parent's DNA fingerprint also.
- determine the genetic diversity within a population. When members of the same population have similar genetic finger prints, the population will have little genetic diversity, hence a smaller gene pool.
- establish whether someone was present at a crime scene, complications: DNA left on another occasion, DNA belongs to a relative, DNA may have been contaminated which affects the action of restriction endonucleases
- Diagnose diseases - such as Huntingdons
- Prevent undesirable breeding in farms and zoos.
- identify a particular allele of a desirable gene - from plants and animals
Extraction: DNA extracted by separating it from the rest of the cell. The quantity of DNA is increased by polymerase chain reaction / PCR.
Digestion: DNA is cut into fragments using restriction endonucleases.
Separation: DNA fragments are separated according to size by gel electrophoresis (and under electrical voltage). Gel is immersed in alkali to separate double strands into single strands. Single strands are transferred to a nylon membrane by southern blotting.
Hybridisation: Complemetary fluorescent DNA probes bind with core sequences. Probes bind under conditions e.g. temp/pH and different probes bind with different core sequences.
Development: An X-ray film is put over the nylon membrane and exposed to radiation from the radioactive probes or positions are located visually if fluorescent probes are used. Because the points correspond to the position of DNA fragments as separated during electrophoresis à a series of bars is revealed.