I've found several of my old study books. I really like this example, because it shows off how easy it has become to identify a suspect. I really like to watch the construction of our body, further and further in detail 
ah and I thought it is great to know how the method works under the microscope.
Sorry if there are any spelling mistakes around the technical terms.
And ms paint ftw.
pre information
Until 1985, meaning until the introduction of the DNA-typecast, it was common within the forensic medicine to go through a large amount of bloodtest to identify possible offenders.
- At first, they used serologic tests, eg the analyze of the AB0-system or the method to clause the rhesus factor.
- Between the 60’s and 70’s the medicine invented methods to clause the proteins-polymorphism inside of the body.
- Finally; the histocompatibility antigens (HLA) were used for the typecast. All of the tests needed huge amounts of samples, which are not always available. Also, in most of the cases these test were not specific enough, to bring up a sure result. In the case of the AB0-system it is easy to understand, same goes for the polymorphism on the tier of proteins. It was not safe enough because too many factors were working during the process, also more proteins were sealed within one analyze.
The find was, that it was very rare to get a good result at the first try and later to find the matching person to the common case. Finally these kinds of analytics were very limited due to the point that the samples were having a high balefulness.
DNA- Fingerprinting
1986; the American scientist Alac Jeffrey’s, Leicester University, wrote a manuscript, in which he was talking about a technique; a new way to identify possible criminal suspects. He called it ‘DNA-Fingerprint’. In his manuscript, he was talking about the areas of the not-coding DNA inside of the genome. He called them ‘VNTR’s’ (=variable number of tandem repeats). To bring it into a picture, imagine ‘little satellites’. These little satellites are built out of short sequences inside of a core. They are multiple amplified in the form of a tandem.
The grade of amplification and the length of the little satellites are varying from being to being. We have heard that the human genome includes about 3 X 10^9 BP. Only 10% of this genetic information coded for proteins and stable RNA (ribunucleic acid). The areas are inside of any race or species very tinned. Including the human being. The rest, about 90% of the genomes of higher eukaryotes are made out of not-coded DNA. These 90% are including, of course, very important and along with that very tinned areas, like the very different and complex control regions in front and behind the genes. The not-coded DNA has still a huge part of DNA in itself. Until know, the meaning of this part is still a mystery. Inside of this area, it is able to find a great difference of sequences, between beings of the same race. A part of this not-coded areas – about 30% of the eukaryotic genomes – are made of reiterirative sequences.
The erratic distributed little satellite uinits are also a part of it.
The core units, from many of these satellites are very similar. The origin of Jeffrey’s method was used to prove the core structures via hybridization inside of the little satellites. The result should be a distinct ‘bar-code’ for every individual DNA.
To proof the existence of the areas he used two DNA-probes. He gave them the names 33.6 and 33.15. Behind these names are the sequences
- 5’-(AGGGCTGGAGG)3-3’, existing 18 times inside of the genome (33.6) and
- 5’-(AGAGGTGGGCAGGTGG)-3’, existing 29 times inside of the genome (33.15)
These little satellites are multi local distributed inside of the genome. The number of the Loci, varies from being to being. His method is today known under the title of ‘multi-locus-probe’ (MLP).
'multi-locus-probe' method MLP
Against this way stands the ‘single-locus-probe’ method. Today preferential used in most of the criminal investigation laboratories.
On the contrary to the MLP, in which method we are getting a huge amount of different sizes of the fragments; the SLP is only showing one or two bonds per hybridization. To identify a being exactly on this polymorphism, we have to use more than one analyze with the SLP method.
THE method
MLP and SLP are variations of the ‘southern-blot-method’. To bring it to work, we are using DNA out of biological parts like blood, semen, hair root or cells out of the buccal mucosa, coming from sputum restenosis. The parts are isolated and cleaned.
It is clear that the DNA-amount, we can achieve out of these samples is not very much. Typically we can isolate a DNA-amount of 500 ng out of a 0,5cm bloodstain.
The cleaned DNA is than digested with a restriction endonuclease.
The standard enzyme of Europe (while working) is therestriction endonuclease Hinf I. It hydrolyzes the sequence 5’-GANTC-3’. The USA on the other hand are having the standard enzyme restriction endonuclease Hae III. Unfortunatelly it is not possible to match the results of different restriction endonucleases with each other.
After the hydrolization, the DNA fragments are separated within an agarosematrix. Separated by the different sizes; to nitrocellulose or nylon membranes. After the transfer is done, they are going to get a hybridization by radioactive samples. Today we are using synthetic oligon nucleotides. It is important that this sequence is matching the core sequence of the little satellites. Even with a short amount of DNA concentration, it is possible to get a result within two days. Thanks to the high sensitive detection methods.
Interpretation of the file
The interpretation of the files is easier. We apply the following samples in a specific order on a gel:
- Samples out of the evidence
- A sample, which includes DNA of the suspect
- A sample, which includes DNA of the victim
The including bond patterns are now matched with each other. Out of experience we know, that the likelihood of matching bonds within the DNA of two not familiar beings can not be higher than 0,25.
- example: Within a MLP analyze we have 11 bonds, so is the likelihood that the second DNA has the same bond profile than the first only about 0,25^11 = 1:4,000,000
MLP analyzes are due to the point of their intricacy very meaningful but in some cases very difficult to interpreted.
The SLP is the exact opposite of the MLP. To get a real result out of a SLP analyze it is important to do more than one SLP analyze; every time with different hydrolyzed DNA-samples. So it is possible to match the new files with the files inside of the database. Not every bond is the same, because there are different DNA-fragments in different populations.
Statistic
There has always been a discussion about it, around scientist and of course lawyers, how relevant, results of DNA fingerprint analyzes are in front of the court. There was the speculation; that specific DNA-profiles, in ethnic subpopulations, are more identically than our filed databases include. This thing was proofed via an experiment. The result was negative. The scientists could not find a higher matching result within the subpopulations than in the random matched populations of the filed files. Obvious, is the area of mutation rates, used within the DNA-Fingerprinting’, much higher. This DNA is not transcribed into functional molecules (proteins or RNA). Because of this, there is no natural selection and the chance for mutations is much higher. Due to this, are these genome areas also members of short, genetic right homogenic populations, polymorph.
Polymerase-Chain-Reaction
A limitation of the DNA-Fingerprinting, by the Southern Blot Method is like always the minimal amount of samples. We can fight the problem today. We are going to bring the polymerase chain reaction as a part to the evidence strategy. This method is very sensitive. A wrong result is nearly impossible.
Summary
Molecular genetic methods are a daily part of modern, forensic medicine. It was possible because these methods are more sensitive than the elder ones. On the other hand are more polymorphism grades in the goal structures included, than the ones, we can find in average structures. At least, the samples are much longer tenable, than 30 years ago. It was possible because modern science made it possible for us to keep the DNA and not only the included proteins. With enough research and complex analyzes the result is in nearly every case reliable. Of course it is possible to create a theoretical case in which it is possible, to receive a matching DNA out of the DNA of another being. If we are going to combine this result with another factor, eg the likelihood the case that a being with a theoretical fingerprint pattern was at this moment seen at a crime scene, makes the thing more unreal. But no matter how clear the results are, there are still some countries that don't except, this kind of proof over a criminal case as the only evidence.
ah and I thought it is great to know how the method works under the microscope. Sorry if there are any spelling mistakes around the technical terms.
And ms paint ftw.
DNA-Characterization in the forensic science.
pre information
Until 1985, meaning until the introduction of the DNA-typecast, it was common within the forensic medicine to go through a large amount of bloodtest to identify possible offenders.
- At first, they used serologic tests, eg the analyze of the AB0-system or the method to clause the rhesus factor.
- Between the 60’s and 70’s the medicine invented methods to clause the proteins-polymorphism inside of the body.
- Finally; the histocompatibility antigens (HLA) were used for the typecast. All of the tests needed huge amounts of samples, which are not always available. Also, in most of the cases these test were not specific enough, to bring up a sure result. In the case of the AB0-system it is easy to understand, same goes for the polymorphism on the tier of proteins. It was not safe enough because too many factors were working during the process, also more proteins were sealed within one analyze.
The find was, that it was very rare to get a good result at the first try and later to find the matching person to the common case. Finally these kinds of analytics were very limited due to the point that the samples were having a high balefulness.
DNA- Fingerprinting
1986; the American scientist Alac Jeffrey’s, Leicester University, wrote a manuscript, in which he was talking about a technique; a new way to identify possible criminal suspects. He called it ‘DNA-Fingerprint’. In his manuscript, he was talking about the areas of the not-coding DNA inside of the genome. He called them ‘VNTR’s’ (=variable number of tandem repeats). To bring it into a picture, imagine ‘little satellites’. These little satellites are built out of short sequences inside of a core. They are multiple amplified in the form of a tandem.
The grade of amplification and the length of the little satellites are varying from being to being. We have heard that the human genome includes about 3 X 10^9 BP. Only 10% of this genetic information coded for proteins and stable RNA (ribunucleic acid). The areas are inside of any race or species very tinned. Including the human being. The rest, about 90% of the genomes of higher eukaryotes are made out of not-coded DNA. These 90% are including, of course, very important and along with that very tinned areas, like the very different and complex control regions in front and behind the genes. The not-coded DNA has still a huge part of DNA in itself. Until know, the meaning of this part is still a mystery. Inside of this area, it is able to find a great difference of sequences, between beings of the same race. A part of this not-coded areas – about 30% of the eukaryotic genomes – are made of reiterirative sequences.
parts of a chromosome, where we can find reiterative sequences
The erratic distributed little satellite uinits are also a part of it.
The core units, from many of these satellites are very similar. The origin of Jeffrey’s method was used to prove the core structures via hybridization inside of the little satellites. The result should be a distinct ‘bar-code’ for every individual DNA.
To proof the existence of the areas he used two DNA-probes. He gave them the names 33.6 and 33.15. Behind these names are the sequences
- 5’-(AGGGCTGGAGG)3-3’, existing 18 times inside of the genome (33.6) and
- 5’-(AGAGGTGGGCAGGTGG)-3’, existing 29 times inside of the genome (33.15)
These little satellites are multi local distributed inside of the genome. The number of the Loci, varies from being to being. His method is today known under the title of ‘multi-locus-probe’ (MLP).
Against this way stands the ‘single-locus-probe’ method. Today preferential used in most of the criminal investigation laboratories.
'single-locus-probe' method SLP
It is easier and more sensitive than the MLP. The results are way better to document and later to file. For this method we are using a single fragment out of a halpoid genome or two fragments out of a dilpoid genome. Inside of this fragment is a sequence area, that is strongly polymorph, meaning its length is different from one being to another being. We get two bonds on the ‘Blot’, if the chromosomes (inside of this area) are heterozygous (which is the common case). An example for such a trial, used with the SLP-analyse, is the trial 3’-alpha-HVR. We can see a hyper variable part in the area of the alpha-globin gene on chromosome number 16. Meanwhile there exists a huge computer databases, which are updated everyday. It is used to match a possible criminal with filed criminals of the base.
It is easier and more sensitive than the MLP. The results are way better to document and later to file. For this method we are using a single fragment out of a halpoid genome or two fragments out of a dilpoid genome. Inside of this fragment is a sequence area, that is strongly polymorph, meaning its length is different from one being to another being. We get two bonds on the ‘Blot’, if the chromosomes (inside of this area) are heterozygous (which is the common case). An example for such a trial, used with the SLP-analyse, is the trial 3’-alpha-HVR. We can see a hyper variable part in the area of the alpha-globin gene on chromosome number 16. Meanwhile there exists a huge computer databases, which are updated everyday. It is used to match a possible criminal with filed criminals of the base.
On the contrary to the MLP, in which method we are getting a huge amount of different sizes of the fragments; the SLP is only showing one or two bonds per hybridization. To identify a being exactly on this polymorphism, we have to use more than one analyze with the SLP method.
THE method
MLP and SLP are variations of the ‘southern-blot-method’. To bring it to work, we are using DNA out of biological parts like blood, semen, hair root or cells out of the buccal mucosa, coming from sputum restenosis. The parts are isolated and cleaned.
It is clear that the DNA-amount, we can achieve out of these samples is not very much. Typically we can isolate a DNA-amount of 500 ng out of a 0,5cm bloodstain.
The cleaned DNA is than digested with a restriction endonuclease.
The standard enzyme of Europe (while working) is therestriction endonuclease Hinf I. It hydrolyzes the sequence 5’-GANTC-3’. The USA on the other hand are having the standard enzyme restriction endonuclease Hae III. Unfortunatelly it is not possible to match the results of different restriction endonucleases with each other.
After the hydrolization, the DNA fragments are separated within an agarosematrix. Separated by the different sizes; to nitrocellulose or nylon membranes. After the transfer is done, they are going to get a hybridization by radioactive samples. Today we are using synthetic oligon nucleotides. It is important that this sequence is matching the core sequence of the little satellites. Even with a short amount of DNA concentration, it is possible to get a result within two days. Thanks to the high sensitive detection methods.
Interpretation of the file
The interpretation of the files is easier. We apply the following samples in a specific order on a gel:
- Samples out of the evidence
- A sample, which includes DNA of the suspect
- A sample, which includes DNA of the victim
The including bond patterns are now matched with each other. Out of experience we know, that the likelihood of matching bonds within the DNA of two not familiar beings can not be higher than 0,25.
- example: Within a MLP analyze we have 11 bonds, so is the likelihood that the second DNA has the same bond profile than the first only about 0,25^11 = 1:4,000,000
MLP analyzes are due to the point of their intricacy very meaningful but in some cases very difficult to interpreted.
The SLP is the exact opposite of the MLP. To get a real result out of a SLP analyze it is important to do more than one SLP analyze; every time with different hydrolyzed DNA-samples. So it is possible to match the new files with the files inside of the database. Not every bond is the same, because there are different DNA-fragments in different populations.
Statistic
There has always been a discussion about it, around scientist and of course lawyers, how relevant, results of DNA fingerprint analyzes are in front of the court. There was the speculation; that specific DNA-profiles, in ethnic subpopulations, are more identically than our filed databases include. This thing was proofed via an experiment. The result was negative. The scientists could not find a higher matching result within the subpopulations than in the random matched populations of the filed files. Obvious, is the area of mutation rates, used within the DNA-Fingerprinting’, much higher. This DNA is not transcribed into functional molecules (proteins or RNA). Because of this, there is no natural selection and the chance for mutations is much higher. Due to this, are these genome areas also members of short, genetic right homogenic populations, polymorph.
Polymerase-Chain-Reaction
A limitation of the DNA-Fingerprinting, by the Southern Blot Method is like always the minimal amount of samples. We can fight the problem today. We are going to bring the polymerase chain reaction as a part to the evidence strategy. This method is very sensitive. A wrong result is nearly impossible.
Summary
Molecular genetic methods are a daily part of modern, forensic medicine. It was possible because these methods are more sensitive than the elder ones. On the other hand are more polymorphism grades in the goal structures included, than the ones, we can find in average structures. At least, the samples are much longer tenable, than 30 years ago. It was possible because modern science made it possible for us to keep the DNA and not only the included proteins. With enough research and complex analyzes the result is in nearly every case reliable. Of course it is possible to create a theoretical case in which it is possible, to receive a matching DNA out of the DNA of another being. If we are going to combine this result with another factor, eg the likelihood the case that a being with a theoretical fingerprint pattern was at this moment seen at a crime scene, makes the thing more unreal. But no matter how clear the results are, there are still some countries that don't except, this kind of proof over a criminal case as the only evidence.
