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Chapter 4. DNA extraction and quantification

 4. DNA extraction and quantification


DNA extraction has two main aims: first, to maximizing the yield of DNA from a sample and in sufficient quantity to permit a full DNA profile to be obtained – this is increasingly important as the sample size diminishes; and, second, to extract DNA that is pure enough for subsequent analysis: the level of difficulty here depends very much on the nature of the sample. Once the DNA has been extracted, quantifying the DNA is important for subsequent analysis.

 


DNA extraction

There are many methods available for extracting DNA. The choice of which method to use depends on a number of factors, including the sample type and quantity; the speed and in some cases ability to automate the extraction procedure [1–5]; the success rate with forensic samples, which is a result of extracting the maximum amount of DNA from a sample and at the same time removing any PCR inhibitors that will prevent successful profiling [2, 6–8]; the chemicals that are used in the extraction – most laboratories go to great lengths to avoid using hazardous chemicals; and the cost of the procedure. Another important factor is the experience of the laboratory staff.

General principles of DNA extraction

    The three stages of DNA extraction can be classified as (i) disruption of the cellular membranes, resulting in cell lysis, (ii) protein denaturation and (iii) the separation of DNA from the denatured protein and other cellular components. Some of the extraction methods commonly used in forensic laboratories are described below.

 


Chelex 100 resin

The Chelex 100 method was one of the first extraction techniques adopted by the forensic community. Chelex 100 is a resin that is composed of styrene– divinylbenzene copolymers containing paired iminodiacetate ions [9]. The resin has a very high affinity for polyvalent metal ions, such as magnesium (Mg2+); it chelates the polyvalent metal ions and effectively removes them from solution.

 


 The extraction procedure is very simple, the Chelex 100 resin, which is supplied as beads, is made into a 5% suspension using distilled water. The cellular material is incubated with the Chelex 100 suspension at 56 ◦C for up to 30 minutes. Proteinase K, which digests most cellular protein, is often added at this point. This incubation is followed by 8–10 minutes at 100 ◦C to ensure that all the cells have ruptured and that the protein has denatured. The tube is then simply centrifuged to pellet the Chelex 100 resin and the denatured protein at the bottom of the tube, leaving the aqueous solution containing the DNA to be used in PCR (Figure 4.1). The Chelex 100 suspension is alkaline, between pH 9.0 and 11.0, and as a result DNA that is isolated using this procedure is single-stranded.

 


DNA EXTRACTION AND QUANTIFICATION

Figure 4.1 The Chelex 100 extraction is quick and easy to perform. (a) The cellular material is added to 1 ml of TE (1 mm EDTA, 10 mm Tris: pH 8.0) and incubated at room temperature for 10–15 minutes. (b) The tube is centrifuged at high speed to pellet the cellular material and the supernatant is removed. (c) The pellet of cellular material is resuspended in 5% Chelex, the tube is incubated at 56 ◦C for 15–30 minutes and then placed in a boiling water bath for 8 minutes. The tube is centrifuged at high speed for 2–3 minutes to pellet precipitated protein. (d) The supernatant contains the DNA and can be used directly in a PCR

 


    The major advantages of this method are it is quick, taking around a hour; it is simple and does not involve the movement of liquid between tubes, thereby reducing the possibility of accidentally mixing samples; the cost is very low; and it avoids the use of harmful chemicals. Importantly, it is amenable to a wide range of forensic samples [9]. The DNA extract produced using this method is relatively crude but sufficiently clean in most cases to generate a DNA profile.

 


Silica-based DNA extraction

    Within molecular biology generally, the ‘salting out’ procedure has been widely used [1]. The first stage of the extraction involves incubating the cellular material in a lysis buffer that contains a detergent along with proteinase K. The commonly used detergents are sodium dodecyl sulphate (SDS), Tween 20, Triton X-100 and Nonidet P-40. The lysis buffer destabilizes the cell membranes, leading to the breakdown of cellular structure.

 


    The addition of a chaotropic salt, for example 6 M guanidine thiocyanate [10] or 6 M sodium chloride, during or after cell lysis, disrupts the protein structure by interfering with hydrogen bonding, Van der Waals interactions and hydrophobic interactions. Cellular proteins are largely insoluble in the presence of the chaotropic agent and can be removed by centrifugation or filtration. The reduced solubility of the cellular protein is caused by the excess of ions in the high concentration of salt competing with the proteins for the aqueous solvent, effectively dehydrating the protein. Commonly used commercial kits, for example the Qiagen kits, exploit the salting-out procedure; the methods to isolate the DNA after the cellular disruption vary widely.

 


    Several DNA extraction methods are based on the binding properties of silica or glass particles. DNA will bind to silica or glass particles with a high affinity in the presence of a chaotropic salt [10, 11]. After the other cellular components have been removed the DNA can be released from the silica/glass particles by suspending them in water (Figure 4.2). In the absence of the chaotropic salt the DNA no longer binds to the silica/glass and is released into solution. The silica method, in particular, has been shown to be robust when extracting DNA from forensic samples [2]; it is also amenable to automation [2–4].

 


    The advantage of the silica-based salting-out methods are that they yield high molecular weight DNA that is cleaner than DNA from Chelex 100 extractions. As with Chelex 100 extractions, no highly toxic chemicals are involved. The process takes longer than the Chelex 100 and involves more than one change of tube and so increases the possibility of sample mixing and cross-contamination.

 

Phenol–chloroform-based DNA extraction

    The phenol–chloroform method has been widely used in molecular biology but has been slowly phased out since the mid-1990s, largely because of the toxic nature of phenol. It is still used in some forensic laboratories; in particular, it is still widely used for the extraction of DNA from bone samples and soils.

 


    Cell lysis is performed as in the previous method. Phenol–chloroform is added to the cell lysate and mixed – the phenol denatures the protein. The extract is then centrifuged and the precipitated protein forms a pellicle at the interface between the organic phenol–chloroform phase and the aqueous phase; this process is repeated two to three times or until there is no visible pellicle [12]. The DNA is then purified from the aqueous phase by ethanol precipitation or filter centrifugation (Figure 4.3). The method produces clean DNA but has some drawbacks: in addition to the toxic nature of phenol, multiple tube changes are required and the process is labour intensive.

 


FTA paper

In Chapter 3 FTA paper was described as a method for sample collection and storage, particularly from buccal swabs and fresh blood samples. Once a sample is applied to the FTA paper it is stable at room temperature for several years. Cellular material lyses on contact with the FTA paper and the DNA becomes bound to the paper, which has been treated with chemicals to inhibit the growth of microorganisms that might otherwise break down the DNA. 

 

DNA EXTRACTION AND QUANTIFICATION

Figure 4.2 DNA extraction from buccal cells using a salting-out method based on the QIAamp Blood Mini Kit. (a) Cellular material is added to a lysis buffer and proteinase K and incubated at 56 ◦C for at least 15 minutes. (b) Ethanol is added to the solution before it is transferred in order to provide the optimum DNA binding conditions. (c) The lysis solution is then transferred to a spin basket that has a membrane that will bind the DNA in the presence of the chaotropic salt. (d) The spin basket is centrifuged and the DNA is captured by the membrane as the solution passes through. (e) Wash buffers are added to the spin basket and (f) pass through the membrane when centrifuged. (g) Typically 100 µl of elution buffer is added to the membrane; in the absence of the chaotropic salt the DNA is released from the membrane and (h) is recovered upon a final centrifugation

 



To analyse the DNA sample, the first step is to take a small region of the card, normally a 2 mm diameter circle, place it into a 1.5 ml tube and the non-DNA components are simply washed off, leaving only DNA on the card. The small circle of FTA paper is then added directly to a PCR (Figure 4.4). The FTA paper extractions are very simple to perform and do not require multiple tube changes, thus reducing the possibility of sample mixing [13–19]. The technology also provides a simple and relatively inexpensive method for long-term storage of DNA, removing the requirement for refrigeration.

 

DNA EXTRACTION FROM CHALLENGING SAMPLES
Figure 4.3 DNA extraction from a buccal swab cells using a salting-out method based on phenolchloroform. (a) Cellular material is added to a lysis buffer and proteinase K and incubated at 56 ◦C for at least 15 minutes. (b and c) The swab is removed and phenol is added, the solution is then vortexed and centrifuged. Precipitated protein and carbohydrate form a pellicle at the interface; this step is repeated until there is no visible material at the interface. Protocols vary – some use only

 



 phenol, others phenol and chloroform (isoamyl alcohol may be added to the phenol/chloroform mixture to prevent it separating). (d) In a final step chloroform alone is added; this removes any residual phenol, which would inhibit downstream processes such as PCR. The aqueous phase now contains DNA. This can be concentrated by adding sodium acetate and either ethanol or iso-propanol to precipitate the DNA, followed by centrifugation (the DNA will precipitate and form a pellet) or by using filter centrifugation, which is similar to the steps in Figure 4.2g– f, except that the membrane acts as a molecular sieve – allowing small molecules to pass through while retaining DNA strands.

DNA extraction from challenging samples

    The extraction of the many samples encountered in the forensic laboratory, including blood and shed epithelial cells, can be carried out routinely using any of the above techniques. There are however some sample types that necessitate variations on the basic techniques.

 



Semen

Semen is one of the most commonly encountered types of biological evidence. The extraction of DNA from the spermatozoa is complicated by the structure of the spermatozoa (Figure 4.5). DNA is found within the head of the spermatozoa that is capped by the protective acrosome, which is rich in the amino acid cysteine; a large number of disulfide bridges form between the cysteine residues in the acrosome. Proteinase K, which is a general proteinase, cannot break the disulfide bonds: however, the addition of dithiothreitol (DTT), a reducing agent that will break the disulfide bonds, greatly increases the release of spermatozoa DNA [20].

 



DNA EXTRACTION AND QUANTIFICATION 

Figure 4.4 DNA extraction from blood on FTA paper. (a) Sections of the FTA card are removed with a punch (usually 1.2 mm or 2 mm diameter), added to FTA purification reagent, mixed and incubated at room temperature for 5 minutes; one or more punched discs can be added to the extraction. (b) The liquid is removed and replaced with fresh purification reagent; this process is repeated two or three times. (c) The discs are then washed two or three times in TE (10 mm Tris-HCl, 0.1 mm EDTA, pH 8.0). (d) Finally, the TE is removed and the FTA discs, containing the DNA, are left to dry at room temperature or with gentle heat (approximately 50 ◦C). The discs can now be added directly to a PCR reaction
 

 


Figure 4.5 The nucleus in the spermatozoa is protected by the acrosome

    Another complication with semen is that it is often recovered as a mixture of spermatozoa and epithelial cells. The acrosome can be an advantage in these cases as it is possible to perform differential lysis: the epithelial cells are broken down by mild lysis conditions and the spermatozoa can be effectively separated from the lysed epithelial cells [20, 21].

 



Hair shafts
    
    Hair shafts that have been pulled out often possess a root that is rich in cellular material and DNA can be extracted using any of the commonly used techniques – plucked roots have been shown to contain as much as 0.5 µg of DNA [22]. Hair that has been shed when it is in the resting telogen phase often contains no cellular material around the root. The hair shafts are composed of keratin, trace metals, air and pigment – cell fragments, including DNA can get trapped in the matrix of the hair and provide enough DNA to produce a profile. However, hair is notoriously difficult to analyse, and in many cases it is only possible to successfully profile mitochondrial DNA [22], although nuclear DNA can, in some cases, be recovered [23].

 



    The hair shaft, like the spermatozoa acrosome, is rich in disulfide bridges and requires either mechanical grinding [24] or the addition of a reducing agent such as DTT [22, 23] that will break the disulfide bonds and allow proteinase K to digest the hair protein and release any trapped nucleic acids. Once released, the DNA can be extracted using the salting-out procedure [25] or organic phenol–chloroform-based extraction [22–24]. Alternative methods include digestion in a buffer containing proteinase K followed by direct PCR [26, 27] or dissolving the hair shaft in sodium hydroxide and, after neutralization, the released DNA is concentrated using filter centrifugation [28].

 


    Because the hair shaft contains very low levels of DNA it is prone to contamination, but unlike many other types of biological evidence with low levels of DNA it is possible to clean the hair shaft prior to DNA extraction. Several methods have been used to clean hair including washing in mild detergents, water and ethanol and also using a mild lysis step in the same way as is used in the differential extraction of semen [29].

 



Hard tissues

    Following murders, terrorist attacks, wars and fatal accidents it is desirable to group together body parts from individuals when fragmentation has occurred and ultimately to identify the deceased. If the time between death and recovery of the body is short then muscle tissues provide a rich source of DNA [30], which can be extracted using, for example, any of the Chelex, salting-out and organic extraction methods. If, however, the soft tissues are displaying an advanced state of decomposition they will not provide any DNA suitable for analysis. When the cellular structure breaks down during decomposition, enzymes that degrade DNA are released and the DNA within the cell is rapidly digested. This process is accelerated by the action of colonizing bacteria, fungi and insects.

 


    Osteocytes are the most common nucleated cells in the bone matrix (Figure 4.6a). In the teeth, odontoblasts within the dentine and fibroblasts in the cell rich zone of the pulp cavity provide a source of nucleated cells [31] (Figure 4.6b). The hard tissues of the body, bone and teeth provide a refuge for DNA. In addition to the physical barriers, the hydroxyapatite/apatite mineral that is a major component of the hard tissues, stabilizes the DNA which becomes closely bound to the positively charged mineral; this interaction limits the action of degrading enzymes [32].

 


    Hard tissues provide an advantage over other forms of biological material because they have a surface that can be cleaned to remove any contaminating DNA using detergents to remove any soft tissue [33], followed by physical abrasion, soaking in sodium hypochlorite (bleach) [34] (Figure 4.7), trypsin enzyme [35] and exposure to strong ultraviolet light.

 



DNA EXTRACTION AND QUANTIFICATION
              
                                        (a)                                                                 (b)

Figure 4.6 Cellular material in bones and teeth. (a) cross-section through a femur: the Haversian canals are surrounded by concentric layers of bone (lamellae); bone cells (osteocytes) occupy lancunae (Image provided by Prof Tim Arnett, Department of Cell & Developmental Biology, University College London, UK). (b) cross-section through a human tooth showing the dentine (D), odontoblast layer (O) and middle part of the dental pulp (M) (Image provided by Dr Marko Vavpotiˇc, Institute of Forensic Medicine, University of Ljubljana, Slovenia) 

          
                                (a)                                          (b)                                      (c)
Figure 4.7 Bone and tooth material can be vigorously cleaned using: (a) abrasion to remove the outer surface and (b) washing in detergent and bleach to remove contaminating materials. (c) Exposure to strong UV light introduces thymine dimers into any contaminating exogenous DNA – preventing amplification during PCR

 



    After cleaning, the bone/tooth material is normally broken down into a powder by drilling [36] or grinding under liquid nitrogen [37]. The resulting material is then decalcified using 0.5 M EDTA, either before or at the same time as cell lysis [38]. The organic phenol–chloroform and the silica binding extraction methods are commonly used to extract the DNA [7, 37–45]. The process of extracting DNA from bone samples takes longer than with any other type of sample. 

 

Quantification of DNA
    
    After extracting DNA an accurate measurement of the amount of DNA and also the quality of the DNA extract is desirable. Adding the optimum amount of DNA to a PCR will produce the best-quality results in the shortest time; adding too much or not enough DNA will result in a profile that is difficult or even impossible to interpret.

 


 This is especially important when profiling forensic samples, when it is very difficult to know the state of preservation of the biological material and also, in many cases, it is difficult to estimate how much cellular material has been collected. It is less important to quantify DNA when using some reference samples – where similar amounts of DNA can be expected to be extracted each time as there are not very many variables. Even so, many laboratories will still quantify the DNA from reference samples as part of their standard analysis. In response to the importance of quantification of samples recovered from the scene of crime, the DNA Advisory Board in the USA adopted rules that made quantification of human DNA mandatory [46].

 


    The quantity of DNA that can be extracted from a sample depends very much on the type of material. Each nucleated cell contains approximately 6 pg of DNA: liquid blood contains 5000−10 000 nucleated blood cells per millilitre; semen contains on average 66 million spermatozoa per millilitre (the average ejaculation produces 2.75 ml of semen) [47]. Biological samples recovered from the scene of crime are not usually in pristine condition and can often consist of a very small number of shed epithelial cells; consequently, the amount of DNA that can be recovered can be extremely low and difficult to quantify.

 

Visualization on agarose gels
    
    A relatively quick and easy method for assessing both the quantity and the quality of extracted DNA is to visualize it on an agarose gel. Agarose is a polymer that can be poured into a variety of gel forms – mini-gels approximately 10 cm long are sufficient to visualize DNA. The gel is submerged in electrophoresis buffer and the DNA solution is loaded into wells that are formed in the gel by a comb; an electric current is applied across the gel and the negatively charged DNA migrates towards the anode. 

 


The agarose gel forms a porous matrix and smaller DNA molecules move through the gel more quickly than do larger DNA molecules. Dyes that intercalate with the DNA double helix, such as ethidium bromide [12], can be added to the gel either before or after electrophoresis, the amount of intercalated dye is proportional to the quantity of DNA. An alternative dye, 4 ,6-diamidino-2-phenylindole (DAPI), can be added directly to the DNA before electrophoresis. This migrates through the gel bound to the minor groove of double-stranded DNA [48]. DNA is visualized by placing the gel on a transilluminator that emits UV light at 260 nm. Quantification standards can be run alongside the unknown samples to allow the DNA concentrations to be estimated. In addition to showing the presence of DNA, the size of the extracted DNA molecules can also be estimated. High molecular weight DNA can be seen as a single band while degraded DNA or DNA that has been sheared during extraction appears as a smear. This makes comparison to the standards difficult as the DNA is spread out over a range a random sizes.

 


    The advantages of agarose gel electrophoresis are that it is quick and easy to carry out and also gives an indication of the size of the DNA molecules that have been extracted. The disadvantages are that quantifications are subjective, based on relative band intensities; it is difficult to gauge the amounts of degraded DNA as there is no suitable reference standard; total DNA is detected that can be a mixture of human and microbial DNA, and this can lead to overestimates of the DNA concentration; the sensitivity of the dye under UV light is poor, so low level DNA will not be visible; it cannot be used to quantify samples extracted using the Chelex method, as this produces single-stranded DNA and the fluorescent dyes that intercalate with the double-stranded DNA do not bind to the single-stranded DNA.
 


Ultraviolet spectrophotometry

    DNA absorbs light maximally at 260 nm. This feature can be used to estimate the amount of DNA in an extract and by measuring a range of wavelengths from 220 nm to 300 nm it is also possible to assess the amount of carbohydrate (maximum absorbance 230 nm) and protein (maximum absorbance 280 nm) that may have coextracted with the sample. The DNA is placed in a quartz cuvette and light is shone through and the absorbance is measured against a standard. A clean DNA extract will produce a curve as shown in Figure 4.8; if the DNA extract is clean, the ratio of the absorbance at 260 nm and 280 nm should be between 1.8 and 2.0 [12].

 


    Spectrophotometry is commonly used for quantification in molecular biology laboratories but has not been widely adopted by the forensic community. The major disadvantage is that it is difficult to quantify small amounts of DNA accurately using spectrophotometry; also, it is not human specific and other chemicals, for example dyes from clothing and humic acids from bone samples, can interfere with the analysis.

Figure 4.8 UV absorbance by a solution containing DNA is maximal at 260 nm. The 260:280 ratio of 1.91 indicates that the extract is not contaminated with protein


Fluorescence spectrophotometry

    Ethidium bromide or DAPI can be used to visualize DNA in agarose gels; these are both examples of chemicals that fluoresce at much higher levels when they intercalate with DNA. In addition to staining agarose gels, fluorescent dyes can also be used as an alternative to UV spectrophotometry for DNA quantitation. A range of dyes has been developed that can be used with fluorescent microplate readers and these are very sensitive. The PicoGreen dye is specific for double-stranded DNA and can detect as little as 25 pg/ml of DNA [46]. When PicoGreen binds to DNA the fluorescence of the dye increases over 1000-fold; ethidium bromide in comparison increases in fluorescence 50–100-fold when it intercalates with double-stranded DNA [46]. PicoGreen is very sensitive and is a powerful technique for quantifying total DNA; it does however have the drawback that it is not human specific.

 
Hybridization

    Hybridization-based quantification methods have been widely used in forensic genetics since the early 1990s, in particular a commercially available kit Quantiblot (Applied Biosystems). Extracted DNA is applied to a positively charged nylon membrane using a slot or dot blot process; the membrane is the exposed to a probe, that is specific to human DNA. A commonly used target was the D17S1 alpha-satellite repeat that is on human chromosome 17 in 500–1000 copies. The probe can be labelled in a number of ways including colorimetric and chemiluminescent.

 


    A series of standards is applied to the membrane, and comparison of the signal from the extracted DNA with the standards allows quantification. The advantage of hybridization-based methods is that the quantification is human specific; agarose gel electrophoresis and spectrophotometry detect total DNA and forensic samples that have been exposed to the environment for any length of time are prone to colonization by bacteria and fungi.

 


    The hybridization systems do suffer from a lack of sensitivity. For samples producing a negative result, in many cases it is still possible to generate a profile after PCR. The analysis of the results is also subjective, leading different operators to come to different conclusions. In addition to the limited sensitivity, the process is labour intensive, taking approximately 2 hours to produce the blot. Hybridization-based methods have been largely replaced by real-time PCR systems.

 
Real-time PCR
    
    When generating a DNA profile, the PCR products are normally analysed at the end point after 28–34 cycles. It is, however, possible to monitor the generation of PCR products as they are generated – real time. 

Figure 4.9 (a) The TaqMan quantification system consists of two PCR primers and an internal probe that hybridizes within the amplified region; (b) as the primer extends it encounters the probe, the 5’ exonuclease activity of the Taq polymerase degrades the probe: the reporter molecule is no longer in proximity to the quencher and fluoresces

 



This was first developed using ethidium bromide: as PCR products are generated in each cycle, more ethidium bromide intercalates with the double-stranded DNA molecule and fluoresces under UV light. The increase in fluorescence can be detected using a suitable ‘camera’ [49]. Increasingly sensitive assays have been developed, such as SYBR Green and the TaqMan system. Using SYBR Green, as PCR products are generated, the dye binds to the double-stranded product and the fluorescence increases. The TaqMan system uses a different approach, with two primers and a probe; the probe is within the region defined by the primers and is labelled on the 5' end with a fluorescent molecule and on the 3' end with a molecule that quenches the fluorescence. As the primers are extended by the Taq polymerase, one of them meets the probe, which is degraded by the polymerase, releasing the probe and the quencher into solution – efficient quenching of the fluorescent molecules only occurs when they are in close proximity on the probe molecule (Figure 4.9).

 


    As more PCR products are generated, more fluorescent molecules are released and the fluorescence from the sample increases (Figure 4.10). Real-time assays are highly sensitive, human specific and are not labour intensive. In addition to detecting the quantity of DNA they have also been designed to detect PCR inhibition, DNA degradation, male-specific DNA (Y chromosome) and mitochondrial DNA [50–61].

 


DNA IQ system

    A novel approach to quantification is used in the commercially available DNA IQ Isolation System (Promega Corporation). The isolation method is based on saltingout and binding to silica: a very specific amount of silica-coated beads is added to the extraction and these bind a maximum amount of DNA; therefore, when the DNA is eluted from the beads the maximum concentration is known. It has the advantage of combining the extraction and quantification steps and can be semi-automated, but has the disadvantage of not being human specific. 

                                                                                                REFERENCES
Figure 4.10 Real-time PCR quantification. The schematic diagram shows the results from four different template concentrations: high (           ), medium (            ), low (            ) and no template (           ). As the PCR cycles the amount of fluorescence generated increases and each sample that contains template will enter an exponential phase; as the reagents become exhausted each reaction will enter a plateau phase. The cycle threshold value (CT) is set to detect the point at which the reaction has entered the exponential phase; the cycle number that this occurs will depend on the amount of template DNA added. To determine the concentration of a DNA extract the CT value is compared to the CT values from a range of standards.

 
References

1. Aljanabi, S.M. and Martinez, I. (1997) Universal and rapid salt-extraction of high quality genomic DNA for PCR-based         techniques. Nucleic Acids Research, 25, 4692–4693. 

2. Castella, V., Dimo-Simonin, N., Brandt-Casadevall, C. and Mangin, P. (2006) Forensic evaluation of the QIAshredder/QIAamp DNA extraction procedure. Forensic Science International, 156, 70–73. 

3. Greenspoon, S.A., Ban, J.D., Sykes, K., Ballard, E.J., Edler, S.S., Baisden, M. et al. (2004) Application of the BioMek (R) 2000 laboratory automation workstation and the DNA IQ (TM) system to the extraction of forensic casework samples. Journal of Forensic Sciences, 49, 29–39. 

4. Montpetit, S.A., Fitch, I.T. and O’Donnell, P.T. (2005) A simple automated instrument for DNA extraction in forensic casework. Journal of Forensic Sciences, 50, 555–563. 

5. Moss, D., Harbison, S.A. and Saul, D.J. (2003) An easily automated, closed-tube forensic DNA extraction procedure using a thermostable proteinase. International Journal of Legal Medicine, 117, 340–349. 

6. Greenspoon, S.A., Scarpetta, M.A., Drayton, M.L. and Turek, S.A. (1998) QIAamp spin columns as a method of DNA isolation for forensic casework. Journal of Forensic Sciences, 43, 1024–1030. 

 



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Chapter 4. DNA extraction and quantification

 4. DNA extraction and quantification DNA extraction has two main aims: first, to maximizing the yield of DNA from a sample and in sufficien...