Human Hair Extraction and Amplification through AP-PCR Procedure Produces Inconclusive DNA Fingerprint

By April Roodbeen

LBS 145

Tuesday 1

Dr. Luckie

4/26/2005

Abstract

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Samples of hair were extracted from the occipital region of the head to obtain DNA to ultimately determine a DNA fingerprint. Arbitrarily Primed Polymerase Chain Reaction (AP-PCR) was then used to amplify the DNA extracted. AP-PCR uses a variety of arbitrary primers (Krha et al., 2004 pg 171). These primers bind to the DNA strands at different sites on the two strands, replicating and amplifying the DNA located in-between the primers (Welsh and McClelland, 1990). AP-PCR produces a variation in the number and sizes of the DNA PCR products (Anonymous-5, Unknown). A number of different primers including DL75â (CAATCGCCCGT- 3â), DL85â (CAGCACCCAC-3â), DL95â (TCACCACGGT-3â), and DL105â (GGATATGCCG-3â) were used in an attempt to provide varying DNA fingerprints.

ÊÊGel-electrophoresis was then used to try to determine the specific DNA fingerprints. During gel-electrophoresis, DNA along with bromophenol blue dye was inserted into wells in an agarose gel and then electricity was run through the gel, resulting in the migration of DNA fragments down the length of the gel. The DNA was compared to a known DNA lambda ladder to determine the sizes of the fragments of DNA and then photographed. It was predicted that the use of various primers would produce varying DNA fingerprints at the end of the experiment. However, none of the trials were successful in producing any visible DNA during gel-electrophoresis, which resulted in the DNA fingerprint being considered inconclusive.

Figure 1. Attempt #1 at a DNA fingerprint of human hair. Lane 1, DL75â (CAATCGCCCGT- 3â) primer; lane 2, DL85â (CAGCACCCAC-3â) primer; lane 3, DL95â (TCACCACGGT-3â) primer; lane 4, DL105â (GGATATGCCG-3â) primer; lane 5, empty; lane 6, DNA lambda ladder; lane 7 and lane 8, empty. No DNA was present in lanes 1-4; however, in lane 6, DNA was present in the DNA lambda ladder used.

Discussion

DNA fingerprinting helps distinguish one individual from another based on their unique DNA sequence that when compared to other individuals unique DNA sequence, through the use of a DNA fingerprint, distinguishes that individual from another (Betsch, 1994). DNA fingerprinting has a variety of common uses, for instance, when trying to determine a childâs paternity, to place a suspect at a scene in a crime scene investigation based on biological evidence, or even to diagnose prenatal and newborn children with inherited diseases (Betsch, 1994). Any sample collected from an individual, whether it is blood, semen, hair, or skin cells can be used in DNA fingerprinting, since an individuals DNA sequence is the same throughout the body (Antler, Unknown).

This experiment, however, focused on hair samples plucked from the occipital region of the head. After DNA was quantified, AP-PCR was used to amplify the amount of DNA, using the primers DL75â (CAATCGCCCGT- 3â), DL85â (CAGCACCCAC-3â), DL95â (TCACCACGGT-3â), and DL105â (GGATATGCCG-3â) in an attempt to give varying fingerprints, and then gel-electrophoresis was used to analyze the DNA. It was predicted that because of the use of different, varying primers, different DNA fingerprints would appear on the gel.

DNA Extraction:

ÊÊÊÊÊÊÊÊÊÊÊ Ten to fifteen hairs were plucked from the occipital region of the head, cut 0.5 cm in length (making sure to include the follicles), and then combined with lysis buffer. The sample was then incubated overnight. However, the lysis buffer was made on March 22, 2005, containing Proteinase K, which serves to purify DNA from contaminating proteins (Anonymous-6, Unknown). Because Proteinase K has the potential to degrade, the degradation might have negatively influenced the DNA sample. Adding more Proteinase K as time went by over the weeks of the experiment might have increased the amount of DNA extracted in the sample. However, DNA was still able to be extracted from the hair sample.

DNA Quantification:

ÊÊÊÊÊÊÊÊÊÊ Next, the DNA extracted was quantified using the spectrometer. Absorbance levels at 260, 280, and 310 were read, using the combination of the DNA and TE buffer in a quartz cuvette. A control of TE buffer in a quartz cuvette was used to zero the machine after each absorbance was read. The absorbance levels were as follows: at 260=.153, at 280 =.102, and at 310 =.056. The concentration of DNA was then expressed by the following formula: (.153-.056)/ (.102-.056) = .097/.046= 2.1. Because a concentration of 2.1 was very close to being within the preferred range of 1.8 and 2.0, this was determined to be a sufficient concentration of DNA with which to perform AP-PCR.

AP-PCR:

ÊÊÊÊÊÊÊÊÊÊÊ There were various attempts at AP-PCR. In the first attempt, the primers DL75â (CAATCGCCCGT- 3â), DL85â (CAGCACCCAC-3â), DL95â (TCACCACGGT-3â), and DL105â (GGATATGCCG-3â) were each ran separately, with only one primer per tube. In the second attempt, they were ran again, one primer to a tube, but in addition, the primers DL75â (CAATCGCCCGT- 3â) and DL85â (CAGCACCCAC-3â) were both put into a tube, and DL95â (TCACCACGGT-3â) and DL105â (GGATATGCCG-3â) were also both put into a tube, for a total of 2 primers per tube. The addition of more than one primer to a tube was done in the hopes that the more primers added to a particular DNA sample, the more chances of the primer matching a stretch of DNA and binding to the DNA and thus showing up in gel-electrophoresis, resulting in a DNA fingerprint. However, this did not prove successful, so in the third attempt at AP-PCR, the tubes once again contained one primer each, exactly like the first attempt. Although, the amount of solution per tube increased from 25 µl to 50 µl, along with the amount of DNA, which increased to 7 µl per tube also (refer to the methods section). This was done because the preset temperatures of the cycles that AP-PCR undergoes in the thermocycler were set to 50 µl of solution. The amount of DNA was increased in an attempt to get DNA to be visible on the gel during gel-electrophoresis, thus resulting in a reproducible DNA fingerprint.

ÊÊÊÊÊÊÊÊÊÊÊ Previous research has indicated that although DNA may have been successfully extracted from hair, that does not necessarily mean that PCR has been successful in the amplification of DNA. This would indicate the presence of some sort of PCR-inhibitor, or something that reduces the efficiency of PCR, in the hair sample (Suenaga and Nakamura, 2004). Melanin, a hair pigment, is a known PCR-inhibitor. Hair-dying also has a negative effect on PCR (Suenaga and Nakamura, 2004). Keratin, located in the hair, is also a strong PCR inhibitor (Di Martino, 2004). It should also be noted that DNA extraction from hair is one of the most difficult methods to perform, because the sample, being really small, results in a very small amount of DNA that can be extracted per hair. The various known PCR inhibitors- dye, keratin, and melanin, also make the already finicky PCR procedure a lot harder to perform accurately (Di Martino, 2004).

Gel-electrophoresis:

ÊÊÊÊÊÊÊÊÊÊÊ Various attempts at gel-electrophoresis were all deemed inconclusive. The first attempt, using 19 µl of DNA, along with 1 µl of bromophenol blue dye, for a total of 20 µl per well did not produce any visible DNA once gel-electrophoresis was preformed. The amount of DNA was decreased in the second attempt to 15 µl with 1 µl of dye, making 16 µl of solution total, due to the possibility that previously, too much DNA was being used. This must not have been the case; however, as once again, no visible DNA was present. The third attempt, with 15 µl of DNA and dye combined, also resulted in no visible DNA. Although, in all cases, the lambda DNA ladder was visible, indicating that it was not the skills of the scientist that were lacking, but the DNA. Because there was not any visible DNA in any of the three trials, the prediction of each primer producing a different, varying DNA fingerprint was not supported.

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There are also a number of other variables that could have negatively affected the PCR process. It is possible that none of the primers used matched the particular DNA sequence, so they were not able to bind to the DNA and amplify during AP-PCR. The use of more primers might help to increase the chance of one of the various primers matching the DNA, resulting in the binding and amplification of DNA during PCR. This would, in turn, produce a DNA fingerprint once gel-electrophoresis occurs. Since PCR itself is a finicky procedure, errors in pipetteing could also have occurred, although precautions were taken to prevent these errors. The presence of air bubbles in the tips of the pipettes may have accidentally altered the amount of a solution added to the PCR cocktail. Also, because no one is perfect when pipetteing, amounts of solution may have been altered by simple human error. Alterations like these may have influenced the PCR process negatively, resulting in the decrease or even absence of DNA amplification, further resulting in the absence of DNA when gel-electrophoresis was performed.ÊÊÊ

ÊÊÊÊÊÊÊÊÊÊÊ Although a DNA fingerprint was not produced during the entirety of this experiment, I do not see the experiment as being a waste of time. If anything, this stream has made me realize that real science fails. Very little, if anything will happen and work out correctly the first time. Through the use of many trials and failures, a person will learn. They must not get frustrated and quit, but take a deep breath and go back and determine what may have gone wrong, and decide how to fix it, or what variables to change. It is a process that requires patience, and in the end, one will come out wiser, even if they were not successful in getting any results.