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DNA Fingerprinting Animation
The chemical structure of everyone's DNA is the same. The only
difference between people (or any animal) is the order of the base
pairs. There are so many millions of base pairs in each person's DNA
that every person has a different sequence.
Using these sequences, every person could be identified solely by the sequence of their base pairs. However, because there are so many millions of base pairs, the task would be very time-consuming. Instead, scientists are able to use a shorter method, because of repeating patterns in DNA.
These patterns do not, however, give an individual "fingerprint," but they are able to determine whether two DNA samples are from the same person, related people, or non-related people. Scientists use a small number of sequences of DNA that are known to vary among individuals a great deal, and analyze those to get a certain probability of a match.
The Southern Blot is one way to analyze the genetic patterns which appear in a person's DNA. Performing a Southern Blot involves:
1. Isolating the DNA in question from the rest of the cellular material in the nucleus. This can be done either chemically, by using a detergent to wash the extra material from the DNA,or mechanically, by applying a large amount of pressure in order to "squeeze out" the DNA.
2. Cutting the DNA into several pieces of different sizes. This is done using one or more restriction enzymes.
3. Sorting the DNA pieces by size. The process by which the size separation, "size fractionation," is done is called gel electrophoresis. The DNA is poured into a gel, such as agarose, and an electrical charge is applied to the gel, with the positive charge at the bottom and the negative charge at the top. Because DNA has a slightly negative charge, the pieces of DNA will be attracted towards the bottom of the gel; the smaller pieces, however, will be able to move more quickly and thus further towards the bottom than the larger pieces. The different-sized pieces of DNA will therefore be separated by size, with the smaller pieces towards the bottom and the larger pieces towards the top.
4. Denaturing the DNA, so that all of the DNA is rendered single-stranded. This can be done either by heating or chemically treating the DNA in the gel.
5. Blotting the DNA. The gel with the size-fractionated DNA is applied to a sheet of nitrocellulose paper, and then baked to permanently attach the DNA to the sheet. The Southern Blot is now ready to be analyzed.
In order to analyze a Southern Blot, a radioactive genetic probe is used in a hybridization reaction with the DNA in question. If an X-ray is taken of the Southern Blot after a radioactive probe has been allowed to bond with the denatured DNA on the paper, only the areas where the radioactive probe binds will show up on the film. This allows researchers to identify, in a particular person's DNA, the occurrence and frequency of the particular genetic pattern contained in the probe.
variable number tandem repeat
A variable number tandem repeats (VNTR) is a short nucleotide sequence ranging from 14 to 100 nucleotides long that is organized into clusters of tandem repeats, usually repeated in the range of between 4 and 40 times per occurrence. Clusters of such repeats are scattered on many chromosomes. Each variant is an allele and they are inherited codominantly.
Coupled with Polymerase chain reactions, VNTRs have been very effective in forensic crime investigations. When VNTRs are cut out, on either side of the sequence, by restriction enzymes and the results are visualized with a gel electrophoresis, a pattern of bands unique to each individual is produced. The number of times that a sequence is repeated varies between different individuals and between maternal and paternal loci of an individual. The likelihood of two individuals having the same band pattern is extremely improbable. Southern blotting is also used to visualize the repeat numbers on the chromosomes. Once the tandem repeat has been found, identification of possible restriction sites on either side of the repeats are carried out. Using restriction enzymes will break the DNA into the repeat sequences plus a little on each end. The number of repeats will determine the length of the fragment of DNA. The repeat sequence itself can be used as a probe, or if the repeat is not long enough, a sequence from the upstream or downstream side can be used. The probe can either be radioactive or have a biotinylated linker for a fluorescent molecule.
In looking at the VNTR data, two basic principles can be relied on:
[b]Tissue Matching[/b]- both VNTR bands must match. If the two samples are from the same individual, he must have exactly the same binding pattern.
[b]Inheritance Matching[/b]- the matching bands must follow the rules of inheritance. In matching an individual with his parents, a person must have one band that matches from each parent. If the relationship is further, such as a grandparent, then the matches must be consistent with the relatedness.
[b]VNTR evidence [/b]is considered to be exclusionary, which means that a mismatch (or no match at all) sample can be excluded from the genetic relationship of the original sample trying to be matched.
There are two principal families of VNTR: minisatellites and microsatelites. The former are sequences of 11-16 bp repeated 1000 times. They are important because they are highly repetitive and dispersed into the genome. In humans, they are present in 60 autosomic loci and can be examined by digesting the DNA and hybriding with a monolocus probe or with another probe derived from a sequence that is common to each locus. The other members of the VNTR family are the microsatellites or STR (short tandem repeats).They are represented by short sequences of 100-200 bp given by the repetition of 1-6 bp sequences. They cannot be digested, so they are amplified by a multiplex PCR. Parental investigation with these kind of markers are non suitable between consanguineous, because electrophoresis profiles will be very similar. So it is possible to examine only one locus. In this way the system is perfect: one allele derives from the mother and the other one from the father. Microsatellites have many uses: they can be used in forensics, genetic variability and parentage studies.
Using these sequences, every person could be identified solely by the sequence of their base pairs. However, because there are so many millions of base pairs, the task would be very time-consuming. Instead, scientists are able to use a shorter method, because of repeating patterns in DNA.
These patterns do not, however, give an individual "fingerprint," but they are able to determine whether two DNA samples are from the same person, related people, or non-related people. Scientists use a small number of sequences of DNA that are known to vary among individuals a great deal, and analyze those to get a certain probability of a match.
The Southern Blot is one way to analyze the genetic patterns which appear in a person's DNA. Performing a Southern Blot involves:
1. Isolating the DNA in question from the rest of the cellular material in the nucleus. This can be done either chemically, by using a detergent to wash the extra material from the DNA,or mechanically, by applying a large amount of pressure in order to "squeeze out" the DNA.
2. Cutting the DNA into several pieces of different sizes. This is done using one or more restriction enzymes.
3. Sorting the DNA pieces by size. The process by which the size separation, "size fractionation," is done is called gel electrophoresis. The DNA is poured into a gel, such as agarose, and an electrical charge is applied to the gel, with the positive charge at the bottom and the negative charge at the top. Because DNA has a slightly negative charge, the pieces of DNA will be attracted towards the bottom of the gel; the smaller pieces, however, will be able to move more quickly and thus further towards the bottom than the larger pieces. The different-sized pieces of DNA will therefore be separated by size, with the smaller pieces towards the bottom and the larger pieces towards the top.
4. Denaturing the DNA, so that all of the DNA is rendered single-stranded. This can be done either by heating or chemically treating the DNA in the gel.
5. Blotting the DNA. The gel with the size-fractionated DNA is applied to a sheet of nitrocellulose paper, and then baked to permanently attach the DNA to the sheet. The Southern Blot is now ready to be analyzed.
In order to analyze a Southern Blot, a radioactive genetic probe is used in a hybridization reaction with the DNA in question. If an X-ray is taken of the Southern Blot after a radioactive probe has been allowed to bond with the denatured DNA on the paper, only the areas where the radioactive probe binds will show up on the film. This allows researchers to identify, in a particular person's DNA, the occurrence and frequency of the particular genetic pattern contained in the probe.
variable number tandem repeat
A variable number tandem repeats (VNTR) is a short nucleotide sequence ranging from 14 to 100 nucleotides long that is organized into clusters of tandem repeats, usually repeated in the range of between 4 and 40 times per occurrence. Clusters of such repeats are scattered on many chromosomes. Each variant is an allele and they are inherited codominantly.
Coupled with Polymerase chain reactions, VNTRs have been very effective in forensic crime investigations. When VNTRs are cut out, on either side of the sequence, by restriction enzymes and the results are visualized with a gel electrophoresis, a pattern of bands unique to each individual is produced. The number of times that a sequence is repeated varies between different individuals and between maternal and paternal loci of an individual. The likelihood of two individuals having the same band pattern is extremely improbable. Southern blotting is also used to visualize the repeat numbers on the chromosomes. Once the tandem repeat has been found, identification of possible restriction sites on either side of the repeats are carried out. Using restriction enzymes will break the DNA into the repeat sequences plus a little on each end. The number of repeats will determine the length of the fragment of DNA. The repeat sequence itself can be used as a probe, or if the repeat is not long enough, a sequence from the upstream or downstream side can be used. The probe can either be radioactive or have a biotinylated linker for a fluorescent molecule.
In looking at the VNTR data, two basic principles can be relied on:
[b]Tissue Matching[/b]- both VNTR bands must match. If the two samples are from the same individual, he must have exactly the same binding pattern.
[b]Inheritance Matching[/b]- the matching bands must follow the rules of inheritance. In matching an individual with his parents, a person must have one band that matches from each parent. If the relationship is further, such as a grandparent, then the matches must be consistent with the relatedness.
[b]VNTR evidence [/b]is considered to be exclusionary, which means that a mismatch (or no match at all) sample can be excluded from the genetic relationship of the original sample trying to be matched.
There are two principal families of VNTR: minisatellites and microsatelites. The former are sequences of 11-16 bp repeated 1000 times. They are important because they are highly repetitive and dispersed into the genome. In humans, they are present in 60 autosomic loci and can be examined by digesting the DNA and hybriding with a monolocus probe or with another probe derived from a sequence that is common to each locus. The other members of the VNTR family are the microsatellites or STR (short tandem repeats).They are represented by short sequences of 100-200 bp given by the repetition of 1-6 bp sequences. They cannot be digested, so they are amplified by a multiplex PCR. Parental investigation with these kind of markers are non suitable between consanguineous, because electrophoresis profiles will be very similar. So it is possible to examine only one locus. In this way the system is perfect: one allele derives from the mother and the other one from the father. Microsatellites have many uses: they can be used in forensics, genetic variability and parentage studies.
