DNA profiling

what are introns

• over 90% of the DNA is made up of the introns: repetitive non coding regions between the genes

• some of these sequences can code for small interfering RNA molecules (siRNA) that interact with mRNA and prevent the production of certain proteins.

• Introns are the regions of the chromosomes that are used in DNA profiling

what are micro-satellites

a section of DNA with a 2-6 base sequence repeated 5 to 100 times

what are mini-satellites

a section of DNA with a 10-100 base sequence repeated 50 to several hundred times

the same mini- and micro-satellites appear in the same positions on each pair of homologous chromosomes. However, the number of repeats of each satellite varies because different patterns will be inherited from each parent

why is pattern of DNA very different

• there are many different introns and a huge variation in the number of repeats so probability that any two individuals have the same pattern of DNA is very unlikely unless they are identical twins

• the more closely related two individuals are, the more likely it is that similarities will be apparent in their DNA patterns

what are recognition sites?

specific base sequences where restriction endonucleases cut the DNA molecule

what is restriction endonucleases

enzymes used to cut up strands of DNA at particular points in the intron sequences

How DNA fragments produced

• the strands of DNA from a sample are cut into fragments using enzyme restriction endonucleases

• these enzymes cut the DNA at particular points in the intron sequences

• there are many different restriction enzymes, each type cutting a DNA moelcule into fragments at different specific base sequences known as recognition sites

• using restriction enzymes that cut either size of mini- and micro-satellite units leaves the repeated sequences intact

• the technique produced a mixture of different-sized DNA fragments, depending on the number of repeated sequences, which consist largely of mini- and micro satellites

what is gel electrophoresis

• a type of chromatography used in analysis of DNA, RNA and proteins from DNA fragments produced in DNA profiles

how to do gel electrophoresis

• An agarose gel plate is created and wells are cut into the gel at one end

• The gel is submerged in a tank containing electrolyte solution; this is a salt solution that conducts electricity. Buffering solutions added to wells to keep pH constant

• The known DNA samples are transferred into the wells using a micropipette, ensuring that a sample of DNA standard is loaded into the first well

  • The purpose of the standard is to produce a set of known results with which to compare any new results 

• Gel contains a dye that binds to the DNA fragments as they move through it. Visible dye also added to DNA samples

• The negative electrode is connected to the end of the plate with the wells and the positive anode is connected at the far end

  • The DNA fragments move towards the anode due to the attraction between the negatively charged phosphates of DNA and the anode

  • The smaller mass / shorter pieces of DNA fragments move faster and therefore further from the wells than the larger fragments

• Probes are then added, after which an X-ray image is taken or UV-light is shone onto the paper producing a pattern of bands which can be compared to the control, or standard, fragments of DNA

use of dye in gel

• the dye binds to the DNA fragments as they move through it. This dye fluoresces when placed under short-wave ultraviolet (UV) light, so it can reveal the band pattern of DNA

• a visible dye is also added to the DNA samples

• It does not bind with the DNA, but moves through the gel slightly faster than the DNA. This visual signal means the current can be turned off before the samples get to the end

what are probes

• Probes are single-stranded DNA sequences that are complementary to the regions of interest; they can be

  • A radioactive label which causes the probes to emit radiation that makes the X-ray film go dark, creating a pattern of dark bands

  • A fluorescent dye which fluoresces when exposed to UV light, creating a pattern of coloured bands

how gel electrophoresis works

• Molecules move through the agar due to the difference in charge across the gel

  • Positively charged molecules will move towards the cathode (negative pole) while negatively charged molecules will move towards the anode (positive pole)

  • DNA is negatively charged due to the phosphate groups and so when placed in an electric field the molecules move towards the anode

• The molecules are separated according to their size / mass 

  • Different sized molecules move through the gel at different rates

  • The tiny pores in the gel allow smaller molecules to move quickly, whereas larger molecules move more slowly

what are short tandem repeats?

These short tandem repeats are a type of non-coding, repeated sequence of bases known as a variable number tandem repeat, or VNTR

consist of micro-satellite regions that are widely used in DNA identification of suspects; statistically, the chance of two people matching on 11 or more sites is so small that it is considered to be reliable evidence in court

how DNA profiling used in forensic science

• In forensice science, to develop a DNA profile in criminal investigations, we use gene probes to identify short tandem repeats

• the DNA profile will be more accurate if more micro-satellites are used

• If only a few regions are analysed then there is a greater chance that closely related individuals will have an identical profile; an analysis of 11 or more sites is considered to be reliable evidence in a law court

how DNA profile used in familial testing? eg to determine genetic relationships

• During a paternity test the DNA profile of a child is compared with a variety of candidates that could be the potential father

• If many bands of the child's DNA profile match with the bands in a paternity candidate's profile, this could indicate that they are the most likely biological father

  • During fertilisation half of the DNA comes from each parent, so a child will share half of their DNA with a parent

  • When comparing DNA profiles the more bands that match between the profiles, the greater the genetic similarity between those individuals and the closer the relationship

• Important for inheritance claims too

• Important in immigration cases. I fperson has immigrated to a country and wants their children to join them they may have to prove this

how DNA profiles are used in the identification and classification of organisms?

• we can use similarities in DNA patterns to identify relationships between individuals and even between species

• scientists are using these to identify and classify different species of animals and plants and to understand the genetic relationships between them

how DNA profiling can also be useful in selective or captive breeding programmes of animals or cultivation of plants

• DNA profiles of the particular organisms can be compared to determine which are genetically the most different from each other

• These organisms will then be crossbred, ensuring that the individuals that breed together are not closely related

  • Breeding between closely related individuals is known as inbreeding, and can cause genetic problems at an individual and population level

    • In individuals there can be an accumulation of harmful recessive alleles that might otherwise have been masked by healthy dominant alleles

    • Inbreeding leads to a smaller gene pool within a population, which can reduce a population's ability to adapt to change

what are DNA barcodes?

• the Consortium for the Barcode of Life (CBOL), the international Barcode of Life projects (IBOL) and the European Consortium for the Barcode of Life (ECBOL) are large groups of scientific organisations that are developing DNA bar coding as a global standard for species identification

• this involves looking at a short genetic sequence from a part of the genome common to particular groups of organisms

• Bar coding will not replace traditional taxonomy, but support it, even possibly developing tools to give results in the field!