Any biological fluid or tissue that contains nucleated cells can be used as a source of DNA. A nucleated cell is any cell that has a nucleus. In blood the white blood cells have nuclei and therefore have DNA, but the red blood cells do not. The sperm cells contain most of the DNA that is in semen, but there are also some epithelial cells, which contain DNA. Much less DNA is obtained from a comparable quantity of semen that lacks sperm cells. In vaginal swabs and in buccal (inner cheek) swabs the epithelial cells have DNA. Urine has some cells in it but not too many. Sometimes enough DNA can be isolated from urine to enable successful typing, but not always.
All specimens containing nucleated cells are handled pretty much the same way in DNA analysis. The specimen is combined with an enzyme called proteinase K, which hydrolyzes the proteins nonspecifically; that is, it catalyzes the breakdown of cellular protein. Proteinase K also digests cell and nuclear membranes, releasing all the contents of the cell and its nucleus into the solution. The result of this process is called a “digest.” Next, it is common to extract DNA from this mixture using a two-phase system of phenol and chloroform. Two-phase means that these two liquids do not mix; when combined, they form two separate layers in a test tube. The two phases and the digested specimen solution are then mixed by shaking, and this causes much of the protein to move into the chloroform layer. The chloroform layer is discarded, while the phenol phase, which contains the DNA, is kept. This extraction process may be repeated two or more times.
The DNA is finally isolated from the phenol phase either by precipitation with ethyl alcohol or filtration using a miniconcentrator. DNA is insoluble in ethyl alcohol, so adding it to a DNA-containing solution causes the DNA to fall out of solution. A miniconcentrator is a small filtration device. The filter retains DNA but allows solutions of phenol, buffer, and so forth to pass through. This device can essentially wash the phenol out of the DNA. The DNA is then recovered in a buffer solution in which it is stable. The miniconcentrator procedure is now common in forensic labs.
There are other ways of processing the proteinase-K digests, such as using special columns lined with materials that adsorb DNA under certain conditions, then release it under other conditions. The mechanism of this adsorption involves oppositely charged molecules attracting one another. A positively charged molecule lining the column will bind DNA. By changing the pH of the buffer solution passing through the column, one can alter the net charges on the column molecules and the DNA so that DNA can be released from the column.
Tuesday, May 17, 2011
Separation, Labeling, and Detection of DNA Segments
Suppose a scientist were looking for a certain sequence pattern in a mixture of DNA fragments of different sizes. The mixture could be separated by electrophoresis on an agarose gel and then fixed on a membrane by Southern blotting. Then, in order to locate the desired DNA sequence pattern on the blot, a single-stranded segment of DNA called a “probe” could be added directly to the blot.
A probe is a single strand of DNA with a sequence complementary to the target strand of DNA. There are methods to easily label the probe—and thus visualize the target sequence that the probe binds with—using radioactive phosphorus-32 (32P) labels. Once the probe is labeled, it can be placed in a solution with the membrane-containing mixture of all the separated DNA fragments. Under controlled conditions the probe will base pair only with the exactly complementary sequence; this process is called “hybridization.” The reason that the probe can base pair (hybridize) with its complementary sequence directly on the membrane is that the Southern blotting process has already denatured the DNA on the membrane, that is, rendered it single stranded.
The excess probe and solution can then be washed away, leaving the membrane with a radioactive probe adhering to one DNA fragment. The scientist can now visualize the target DNA sequence by placing this membrane against a sheet of X-ray film. The radioactivity exposes the film just as X-rays would. When the film is developed, it shows an image of the DNA fragment that had the radioactive label on it. This process, illustrated in the diagram, is called “autoradiography.” The first forensic DNA typing was done in this way. But radioactive materials such as 32P pose a health hazard, so it is expensive and cumbersome to handle and store them in the safe, legal way. Later, a method using light-emitting (chemiluminescent) probes was developed so scientists could avoid having to work with the radioactive phosphorus.
As already discussed, scientists can separate DNA fragments of different sizes using electrophoresis on agarose gels, but the electrophoresis can also be carried out in tiny capillaries that are filled with a solution of buffer and polymers (somewhat comparable to agarose). This process is capillary electrophoresis (CE) and has some significant advantages over traditional gel electrophoresis. One is that heat does not build up as much in the capillary. Heat buildup is a problem in electrophoresis, both because it can denature protein or DNA molecules and because it can change the properties of the medium (the agarose or buffer solution) in a way that affects the migration properties of the molecules. If heat buildup is not controlled, it can affect the reproducibility of electrophoresis.
DNA probes and Southern blotting. Probe:
Short sequences of single-stranded DNA, 20-50 nucleotides (building blocks) long, can be chemically synthesized and them made radioactive or fluorescent. This DNA can base pair with a complementary DNA or RNA strand, labeling it as either radioactive or luminescent. Southern blotting:
1. The DNA is fragmented by the addition of a restriction enzyme (biological catalyst that cuts DNA at specific sites).
2. The fragments are next separated, according to their length, into invisible bands on electrophoresis gel.
3. The separated DNA fragments can then be transferred to a nitrocellulose or nylon
membrane. The DNA molecules are made to stick permanently to the membrane.
4. When a radioactive DNA probe solution is added to the membrane and then gently washed off, some of the probes will remain attached to any DNA fragments that have a complementary sequence.
5. The membrane is placed against a photographic film. 6. The film darkens wherever the radioactive DNA probe has bound to the membrane. The probe has identified which DNA fragment contains the complementary DNA sequence for the probe.
A probe is a single strand of DNA with a sequence complementary to the target strand of DNA. There are methods to easily label the probe—and thus visualize the target sequence that the probe binds with—using radioactive phosphorus-32 (32P) labels. Once the probe is labeled, it can be placed in a solution with the membrane-containing mixture of all the separated DNA fragments. Under controlled conditions the probe will base pair only with the exactly complementary sequence; this process is called “hybridization.” The reason that the probe can base pair (hybridize) with its complementary sequence directly on the membrane is that the Southern blotting process has already denatured the DNA on the membrane, that is, rendered it single stranded.
The excess probe and solution can then be washed away, leaving the membrane with a radioactive probe adhering to one DNA fragment. The scientist can now visualize the target DNA sequence by placing this membrane against a sheet of X-ray film. The radioactivity exposes the film just as X-rays would. When the film is developed, it shows an image of the DNA fragment that had the radioactive label on it. This process, illustrated in the diagram, is called “autoradiography.” The first forensic DNA typing was done in this way. But radioactive materials such as 32P pose a health hazard, so it is expensive and cumbersome to handle and store them in the safe, legal way. Later, a method using light-emitting (chemiluminescent) probes was developed so scientists could avoid having to work with the radioactive phosphorus.
As already discussed, scientists can separate DNA fragments of different sizes using electrophoresis on agarose gels, but the electrophoresis can also be carried out in tiny capillaries that are filled with a solution of buffer and polymers (somewhat comparable to agarose). This process is capillary electrophoresis (CE) and has some significant advantages over traditional gel electrophoresis. One is that heat does not build up as much in the capillary. Heat buildup is a problem in electrophoresis, both because it can denature protein or DNA molecules and because it can change the properties of the medium (the agarose or buffer solution) in a way that affects the migration properties of the molecules. If heat buildup is not controlled, it can affect the reproducibility of electrophoresis.
Short sequences of single-stranded DNA, 20-50 nucleotides (building blocks) long, can be chemically synthesized and them made radioactive or fluorescent. This DNA can base pair with a complementary DNA or RNA strand, labeling it as either radioactive or luminescent. Southern blotting:
1. The DNA is fragmented by the addition of a restriction enzyme (biological catalyst that cuts DNA at specific sites).
2. The fragments are next separated, according to their length, into invisible bands on electrophoresis gel.
3. The separated DNA fragments can then be transferred to a nitrocellulose or nylon
membrane. The DNA molecules are made to stick permanently to the membrane.
4. When a radioactive DNA probe solution is added to the membrane and then gently washed off, some of the probes will remain attached to any DNA fragments that have a complementary sequence.
5. The membrane is placed against a photographic film. 6. The film darkens wherever the radioactive DNA probe has bound to the membrane. The probe has identified which DNA fragment contains the complementary DNA sequence for the probe.
Tuesday, April 26, 2011
Southern Blots
As DNA analysis methods were being developed, it was common to separate DNA fragments of different sizes on agarose gels, as described in the preceding section. Many times, though, scientists wanted to do further testing steps that just would not work in gels. A biochemist named Edwin Southern came up with a method to solve this problem in 1975.
Southern’s method employs a membrane made of nitrocellulose or nylon, which is laid onto the gel after the DNA fragments have been separated. The gel and membrane are then placed into a buffer solution (a solution that resists change in pH), and some absorbent material is placed on top of the membrane. The absorbent material draws the solution upward, through the gel and through the membrane, but the DNA fragments in the gel are too big to go through the membrane, so they stick to it. Because the membrane is the same size as the gel, it retains the original position and orientation of the DNA fragments in the gel.
Once the DNA fragments are on the membrane, they are stable and cannot easily be removed. Furthermore, the membrane is tough and resilient, allowing scientists to do further tests on the DNA right on the membrane. This process of transferring DNA fragments from a gel to a membrane is called “Southern blotting,” and the resulting membrane is often just called a “blot.” This procedure was an important part of the earliest kind of forensic DNA typing, and it is still used regularly in research labs.
Southern’s method employs a membrane made of nitrocellulose or nylon, which is laid onto the gel after the DNA fragments have been separated. The gel and membrane are then placed into a buffer solution (a solution that resists change in pH), and some absorbent material is placed on top of the membrane. The absorbent material draws the solution upward, through the gel and through the membrane, but the DNA fragments in the gel are too big to go through the membrane, so they stick to it. Because the membrane is the same size as the gel, it retains the original position and orientation of the DNA fragments in the gel.
Once the DNA fragments are on the membrane, they are stable and cannot easily be removed. Furthermore, the membrane is tough and resilient, allowing scientists to do further tests on the DNA right on the membrane. This process of transferring DNA fragments from a gel to a membrane is called “Southern blotting,” and the resulting membrane is often just called a “blot.” This procedure was an important part of the earliest kind of forensic DNA typing, and it is still used regularly in research labs.
Monday, April 25, 2011
Gel Methods
Scientists often need to see whether DNA was successfully prepared from cells or tissues in evidence, how big the molecules are, and how much DNA is in the preparation. Gel methods are commonly used for these purposes. Gel methods provide a way of visualizing DNA.
DNA is a very large molecule that is highly negatively charged. As a result it can be moved by electrophoresis in a gel medium. Electrophoresis is a procedure that uses an electronic field to move big, charged molecules. Agarose gels (similar in consistency to Jell-O) provide a good medium for DNA movement in an electric field. An electrophoresis setup consists of the agarose gel placed in a chamber that separates a positive from a negative compartment and is filled with a solution containing charged particles. The diagram on page 33 shows an electrophoresis setup. A power supply sets up a current in the gel. DNA specimens placed in wells near the negative end will migrate toward the positive end, because DNA is very negatively charged and because unlike charges attract.
Different sized DNA molecules travel different speeds and distances. The larger the DNA molecule, the more slowly it migrates. Thus, the distance a DNA molecule travels gives an indication of how big it is. To help in these estimates scientists typically add standards and calibrators (proteins of known size) to the gel and run them along with the specimens. One factor affecting the size of DNA fragments is degradation. Environmental exposure can cause the DNA in a forensic specimen to degrade, to break into smaller fragments. As a result the specimen will contain an array of different sized DNA molecules. Whereas intact, undegraded DNA appears as a tight band on the agarose gel, degraded DNA is visible as a streak.
DNA is not, however, visible by itself, in a test tube, or on a gel. One must add another material to the DNA to make it visible, and certain dyes that can tuck themselves into the folds of the double helix are used for this purpose. When the DNA-dye complex is illuminated by ultraviolet (UV) light, the DNA fluoresces, so it can be seen. The gels themselves change and degrade over time, so they cannot be kept and stored. Consequently, lab scientists regularly take pictures of these UV illuminated gels to make a permanent record of the outcome of electrophoresis. Furthermore, photography can actually improve visibility of the results. The fluorescent DNA appears white on an otherwise black background in the photo, and it is sometimes easier to see things in the picture than on the gel itself.
DNA is a very large molecule that is highly negatively charged. As a result it can be moved by electrophoresis in a gel medium. Electrophoresis is a procedure that uses an electronic field to move big, charged molecules. Agarose gels (similar in consistency to Jell-O) provide a good medium for DNA movement in an electric field. An electrophoresis setup consists of the agarose gel placed in a chamber that separates a positive from a negative compartment and is filled with a solution containing charged particles. The diagram on page 33 shows an electrophoresis setup. A power supply sets up a current in the gel. DNA specimens placed in wells near the negative end will migrate toward the positive end, because DNA is very negatively charged and because unlike charges attract.
Different sized DNA molecules travel different speeds and distances. The larger the DNA molecule, the more slowly it migrates. Thus, the distance a DNA molecule travels gives an indication of how big it is. To help in these estimates scientists typically add standards and calibrators (proteins of known size) to the gel and run them along with the specimens. One factor affecting the size of DNA fragments is degradation. Environmental exposure can cause the DNA in a forensic specimen to degrade, to break into smaller fragments. As a result the specimen will contain an array of different sized DNA molecules. Whereas intact, undegraded DNA appears as a tight band on the agarose gel, degraded DNA is visible as a streak.
DNA is not, however, visible by itself, in a test tube, or on a gel. One must add another material to the DNA to make it visible, and certain dyes that can tuck themselves into the folds of the double helix are used for this purpose. When the DNA-dye complex is illuminated by ultraviolet (UV) light, the DNA fluoresces, so it can be seen. The gels themselves change and degrade over time, so they cannot be kept and stored. Consequently, lab scientists regularly take pictures of these UV illuminated gels to make a permanent record of the outcome of electrophoresis. Furthermore, photography can actually improve visibility of the results. The fluorescent DNA appears white on an otherwise black background in the photo, and it is sometimes easier to see things in the picture than on the gel itself.
Sunday, April 24, 2011
Restriction Enzymes
Restriction enzymes were important in the RFLP forensic DNA-typing procedure. Many different bacteria make special enzymes called restriction endonucleases, or restriction enzymes. There are many different restriction enzymes, but they all do one thing: They cut the doublestranded DNA molecule at a specific place. Strictly speaking, the enzyme itself does not “cut” DNA; it catalyzes the hydrolysis of DNA. Hydrolysis means that a molecule is separated into two parts and a molecule of water (H2O) is added in the process. But, as a kind of shorthand, molecular biologists often talk about restriction enzymes “cutting” DNA.
A restriction enzyme cuts DNA at a specific place along the chain because it recognizes a short base sequence of four to seven bases. The restriction enzyme HaeIII, for example, recognizes the sequence . . . GGCC . . . and EcoR1 recognizes the sequence . . . GAATTC. Four additional examples of restriction endonucleases are illustrated in the figure. The heavy arrows in the figure show the cut points in double-stranded DNA at the recognition sequence. The earliest forensic DNA-typing method involved the use of restriction enzymes.
A restriction enzyme cuts DNA at a specific place along the chain because it recognizes a short base sequence of four to seven bases. The restriction enzyme HaeIII, for example, recognizes the sequence . . . GGCC . . . and EcoR1 recognizes the sequence . . . GAATTC. Four additional examples of restriction endonucleases are illustrated in the figure. The heavy arrows in the figure show the cut points in double-stranded DNA at the recognition sequence. The earliest forensic DNA-typing method involved the use of restriction enzymes.
Saturday, April 23, 2011
Mitochondrial DNA Inheritance
Because mitochondria have their own DNA, separate from nuclear DNA, one can speak of a “mitochondrial genome,” all the DNA contained in a mitochondrion. Cells have many mitochondria, so there are multiple copies of mtDNA in every cell. The MtDNA genome is significantly smaller than the nuclear genome. The nucleus has about 3.5 billion base pairs in its DNA; the mitochondrion has about 16,500 base pairs in its DNA.
All the variability between people that is forensically useful can essentially be seen by looking through a couple of short sequences in the hypervariable regions. In practice, forensic scientists copy the sequences of interest using the polymerase chain reaction and then analyze the copies. Unlike nuclear DNA typing, which consists of determining the sizes of tandem-repeat regions, mtDNA analysis is sequencing. The variability from person to person in mtDNA consists of a few variations in the base sequence. Thus, the PCR products (the copies) from the hypervariable regions have to be sequenced (the order of the bases in the DNA strand has to be determined).
All the variability between people that is forensically useful can essentially be seen by looking through a couple of short sequences in the hypervariable regions. In practice, forensic scientists copy the sequences of interest using the polymerase chain reaction and then analyze the copies. Unlike nuclear DNA typing, which consists of determining the sizes of tandem-repeat regions, mtDNA analysis is sequencing. The variability from person to person in mtDNA consists of a few variations in the base sequence. Thus, the PCR products (the copies) from the hypervariable regions have to be sequenced (the order of the bases in the DNA strand has to be determined).
Friday, April 22, 2011
Mitochondria and Mitochondrial DNA
The DNA discussed so far is found in the nucleus of the cell, in the chromosomes. In addition to this nuclear DNA, cells also contain DNA in small structures outside the nucleus called “mitochondria.” The accompanying diagram of a generalized animal cell shows the nucleus and mitochondria, as well as the other cell structures. Mitochondria contain the cell’s energy-processing machinery, but they also contain a small amount of DNA. Mitochondrial DNA (mtDNA) is inherited entirely from one’s mother. There is no paternal contribution.
Mitochondrial DNA, unlike its nuclear counterpart, is circular. In that respect it is similar to some bacterial genomes, which consist of a single circular DNA molecule. It is now known that over long periods of time mtDNA occasionally undergoes mutations and that these mutations are stable and passed along from mother to offspring. Anthropologists, scientists who study the variation and evolution of human beings, use mtDNA to follow patterns of human migration over time. Some of the mtDNA codes for specific proteins, but mtDNA also has a region (called the “control region,” or “D-loop”) that, like some sections of nuclear DNA, is subject to a great deal of polymorphism. It can be divided into two hypervariable regions, designated HV1 and HV2, which are 342 and 268 base pairs (bp) in length, respectively. Hypervariable means that these regions are especially prone to random mutations over time, and most of the variability in mtDNA from person to person is found here.
Forensic mtDNA typing is sometimes used in cases when nuclear DNA typing fails or cannot be done. It is also used in trying to identify human remains. Mitochondrial DNA is quite robust in some tissues, especially older or weathered specimens such as old bones. That is the basis for its use in the identification of skeletal remains, which include no soft tissues (and hence no nuclear DNA). In addition, hair shafts contain mtDNA but no nuclear DNA. (Hair roots have nuclear DNA, but many hairs found as evidence have no roots. They are shed from the human body.) So, any DNA analysis on hair shafts must be mtDNA typing.
Mitochondrial DNA, unlike its nuclear counterpart, is circular. In that respect it is similar to some bacterial genomes, which consist of a single circular DNA molecule. It is now known that over long periods of time mtDNA occasionally undergoes mutations and that these mutations are stable and passed along from mother to offspring. Anthropologists, scientists who study the variation and evolution of human beings, use mtDNA to follow patterns of human migration over time. Some of the mtDNA codes for specific proteins, but mtDNA also has a region (called the “control region,” or “D-loop”) that, like some sections of nuclear DNA, is subject to a great deal of polymorphism. It can be divided into two hypervariable regions, designated HV1 and HV2, which are 342 and 268 base pairs (bp) in length, respectively. Hypervariable means that these regions are especially prone to random mutations over time, and most of the variability in mtDNA from person to person is found here.
Forensic mtDNA typing is sometimes used in cases when nuclear DNA typing fails or cannot be done. It is also used in trying to identify human remains. Mitochondrial DNA is quite robust in some tissues, especially older or weathered specimens such as old bones. That is the basis for its use in the identification of skeletal remains, which include no soft tissues (and hence no nuclear DNA). In addition, hair shafts contain mtDNA but no nuclear DNA. (Hair roots have nuclear DNA, but many hairs found as evidence have no roots. They are shed from the human body.) So, any DNA analysis on hair shafts must be mtDNA typing.
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