DNA Profiling Hoza, A. S BLS 209

DNA Fingerprinting (DNA Profiling)

• Makes use of segments of DNA that do not code for protein products,

• • but do exhibit variability (caused by mutation) in the nucleotide base sequences from individual to individual.

• Because the DNA is not involved in making important protein molecules,

• • mutations have no affect on the health or survival of the individual.

• In some instances the segment of DNA being investigated contains varying numbers of repeated letters from one individual to another (i.e molecular stutter).

• One segment of DNA on chromosome #5 that is routinely used in DNA profiling includes from 7 to 15 repeated sequences of the short sequence, AGAT.

• DNA profiling is a highly standardized process.

• E.g FBI has set the standard and routinely uses 13 specific DNA locations on chromosomes 2, 3, 4, 5 (2 loci), 7, 8, 11, 12, 13 16, 18, and 21, in addition to the sex chromosomes, for DNA profiles for humans.

• Each person inherits two versions of these variable length DNA segments on their chromosomes, one from her/his father and one from mother.

• In DNA profiling, these segments of DNA are amplified by PCR, to produce 28 types of DNA fragments of various lengths that are unique to each individual.

• There is one type of fragment from each of the 13 loci from the paternal chromosomes and 13 from the maternal chromosomes, plus the two sex chromosomes.

• Special single-stranded DNA primers that initiate DNA replication just upstream and downstream from the specific DNA fragments insure that only the desired segments of DNA are amplified.

• Each primer is about 25 nucleotides long.

• One of the two primers for each locus is "tagged" with a colored molecule that helps identify the DNA segment that it helps amplify.

• After the DNA segments from a sample of DNA from a person are amplified and "tagged" with the colored primers they are "loaded" onto a gel electrophoresis apparatus and separated by length.

• The DNA fragments are scanned by a laser probe as they pass through the electrophoresis gel.

• The colored primers attached to each fragment are detected as they pass the laser probe.

• Because each person has a different combination of DNA fragments of varying length, the pattern of colors detected by the laser probe is different for each person.

Some Applications of DNA Fingerprinting

• DNA Sequencing:

• • DNA fingerprinting or profiling can be used to identify an individual based on his or her DNA

• • DNA Sequencing can be used to identify an individual,

• • • determine if an individual carries a particular mutation, or

• • • determine the sequence of the entire human genome.

• Eg. Techniques:

• • Cycle sequencing" and DNA "chips

• • • used to determine the specific sequence of nucleotides in the sample to be analyzed.

• Gene Chips - DNA Chips - Biochips Microarrays

• A DNA microarray:

• • A small glass slide upon which from a few to thousands of known samples of single stranded DNA are placed in very, small dots.

• • The type of DNA in each dot is mapped in a computer program

• Samples of DNA to be tested are labeled with a colored dye and applied to the known samples on the chip to see if any of the DNA in the sample matches up with and sticks to the DNA on the chip.

• Scanning the chip through a microscope and comparing the location of the colored dots with the computer map of the sample on the chip indicates which DNA is present in the sample.

The chips may be used to:

• Conduct genetic tests for the presence of a known mutation of a gene.

• Determine which genes are active in a particular organ or in the presence of a particular environmental stimulus:

• • hormone, light, nutrient, etc.

• Look for the presence of a particular organism or genetic material:

• • testing for HIV genetic material, or

• • testing a soil sample for a particular bacterial species.

• Sequence DNA

Using Microarrays to determine which genes are active in a tissue sample:

• Samples of DNA from:

• • part of the genome or

• • every known gene sequence

• • applied to the surface of a glass slide in a series of microscopic dots - an array.

• • When a gene is activated it produces messenger RNA (mRNA) in the process of constructing the protein encoded in the gene.

• • Messenger RNA can be specifically extracted from cells in certain tissues and/or at different times of development.

• • The question to be answered is , "Which specific allele or mutated form of a gene or group of genes is/are present? OR Which genes are active in the cells?

• • The goal of microarray analysis is to match each type of mRNA extracted from a cell with the DNA from the gene that produced it.

• • The chip is then examined under a fluorescence microscope with laser light and a photograph is taken to record the fluorescence of dots on the chip that have hybridized with cDNA prepared from the cell sample .

• • The location of each and every segment of DNA on the chip is known, so determining which genes were active in the cell is just a matter of comparing the pattern of fluorescing dots to the gene map of the chip.