DNA Isolation System for DNA Profiling

1. Introduction

DNA profiling has reached great heights in forensic science with new technological advancements every other year, which makes it possible to associate the evidence with a particular person. DNA profiling techniques are widely used in human identity testing for identifying the suspect, paternity testing, identification of victim’s in mass disaster and identification of a person’s identity from human remains. DNA profiling technology is not only used in human identity testing but also in wild life and conservation genetics.

DNA profiling is most often subjected to discrepancy and number of factors; like the extraction protocol followed, source of the DNA (blood, saliva, semen, skin cells, sweat, hair etc), contamination of the sample, match probability, Partial DNA profile, Low copy number, mixtures, etc can cause ambiguity and the defence counsel often tend to raise questions on these issues .

DNA profiling methods utilize the genotypic differences between the individuals to identify the contributor. Profiling the entire genome is a daunting task to perform, hence for this reason: short tandem repeats on the non coding regions of the genome are profiled. These tandem repeats on specific alleles of the non coding regions are said to be unique among individuals, perhaps the DNA profile as evidence may not be declared as a 100 % match with the profile of the suspect hence forth, a random match probability that some other person unrelated to the suspect having the same profile is calculated.

Finding a suitable DNA isolation system to attain a good profile is important. The extraction procedure varies according to the type of biological evidence and also on the quantity of the evidence collected. The method of choice in a forensic laboratory is one which can yield sufficient quantity of amplified DNA and a good quality profile.

2. Source of DNA evidence:

The efficiency of obtaining a full good quality profile also depends on the source of the DNA sample. Though DNA can be obtained from various sources like Semen, Blood, Hair, Bones, Teeth, saliva, Sweat, Urine and skin cells, but the focus is mainly imposed on the DNA obtained from blood and saliva and buccal swabs when collecting reference samples from a suspect or victim. Chelex and Qiagen are best suited for samples obtained from buccal swabs, skin cells and blood.

Each nucleated cell has approximately 6pg of DNA, while liquid blood has 5000-10,000 nucleated blood cells per/ml. Blood is an excellent source of human DNA. DNA is present in white blood cells of humans, but not in red blood cells which lack nuclei. A small spot of blood, approximately 50 µl is sufficient enough for PCR amplification.

Buccal swabs are easy and a painless method to collect DNA sample. They contain numerous cheek cells which shed very frequently, which is a rich source of cellular DNA. Buccal Swabs also contain considerable amounts of saliva in it, making it even a good source of DNA.

3. Methods for DNA extraction:

The organic extraction methods like phenol chloroform method are cheap and efficient enough to remove PCR inhibitors but greatly reduce the amount of DNA. The other widely used techniques below are used in many forensic laboratories in UK and also in other parts of the world.

3.1 Chelex method:

The Chelex is one of the easiest and simplest methods to use for forensic casework. It works effectively well when the sample quantity is minimal even a “speck of blood”. The extraction mechanism is simple and effective based on cell lysis during heating and binding of Chelex resin (Styrene vinyl benzene copolymers) to the Mg2+ ions preventing the degradation of DNA from DNase yielding a single stranded DNA which is compatible for further PCR analysis.

3.1.1 Advantages:

• Cheap, reliable and fast.
• Less number of sample transfer between tubes , less chance of contamination
• Non hazardous chemicals used
• Yields single stranded DNA, hence compatible with PCR technique.
• Efficient even when the sample amount is slightly minimal than required.

3.1.2 Limitations of Chelex :

• One of the major disadvantages of Chelex is that it is not efficient in removal of inhibitors.
• Not suitable for long term storage because the resin loses its binding capacity with the metal ions.
• Presence of Chelex resin particles even after removal step may sometimes inhibit PCR process.
• Degraded DNA is unsuitable for extraction using the Chelex method because the heating may cause disruptions in the degraded DNA.
• Uneven distribution of resin beads may also affect the PCR process.

3.2 Qiagen:

The QIAamp extraction kit is much more rapid and fast method when compared with the Chelex and other organic extraction methods. The QIAamp kit uses a special spin column which made of a silica gel membrane. Under high salt (Chaotropic) concentrations, the DNA binds with the silica membrane and Cells are lysed on addition of Proteinase, and on further washing and spinning removes unwanted contaminants and inhibitors, while the DNA is still adsorbed to the membrane. The Adsorbed DNA can be finally eluted by rehydration with aqueous low salt solutions. The eluted DNA is double stranded.

3.2.1. Advantages:

• No toxic chemicals used
• High quality yields
• Efficient removal of contaminants and inhibitors
• Can be used for variety of samples like fresh & frozen blood, bone marrow, Viral DNA and saliva and other body fluids.
• Sample size of about 200 µl is sufficient enough

3.2.2 Disadvantages:

• Frequent Transfer of spin columns
• The DNA sometimes is not eluted properly from the silica membrane.
• Or the DNA is washed away during the washing procedure.

4. Q-PCR techniques:

Determining the amount of DNA in a sample is essential for PCR. Adding right amount of DNA to the PCR amplification is the primary essential step for obtaining a good quality profile because PCR reaction is very sensitive, too much DNA to the reaction can cause over amplification while minimal amounts results in a poor profile. Both the methods mentioned below works on the basic mechanism of PCR.

PCR, the amplicons are designed in such a way that the smaller ones gets amplifies preferentially because the STR allelic remain as the target

4.1 The home brew methods:

Home brew methods are reliable and inexpensive but at the same time it is time consuming because the preparation and addition of each component of the mixture is done manually. There are high chances of contamination; hence extra quality control measures should be taken. One of the major drawbacks of home brew based PCR methods is that the positive control is added externally. Moreover the primers designed are sometimes not compatible with the sequences in the allelic population.

4.2 PLEXOR:

Plexor is commercially made available by the ‘Promega’ company. The advantage of Plexor with other “Home brew” methods is it is highly sensitive and it can quantify both autosomal human DNA and also the male specific DNA simultaneously for samples of even 3 pg/ul of DNA. Plexor measures the decrease in fluorescence of the dye as the PCR product is synthesized unlike the other home brew Q-PCR methods where, increase in fluorescence is detected. Real time software such as the Plexor Analysis software detects and analyzes the fluorescence data. The kit also contains internal positive and negative controls unlike the home brew PCR methods where, the controls are added manually to the mixture. Three different dyes are used for quantification ,the FAM dye is used to detect the human autosomal DNA , CAL FLUOR ORANGE 560 dye is used to detect the Y – chromosomal DNA, CAL FLUOR ORANGE 610 dye used to detect Internal PCR control while , IC5 dye used as the reference dye.

4 .PCR AMPLIFICATION:

The PCR reaction allows specific amplification of DNA sequence of interest, in a forensic case work the tandem repeated allele sequences are targeted and amplified. The success of the PCR is based on the selection of the primers in context of its use. The result is a calibration curve which shows the concentration of DNA in each of the tube against the standard curve.

5.1 Powerplex:

The Powerplex 16 kit amplifies alleles in the loci Penta E, D18S51, D21S11, TH01, D2S1358, FGA, TPOX, D8S1179, vWA, Amelogenin, PentaD, CSF1PO, D16S39, D7S820, D13S317 and D5S818. The alleles are differentiated from one another by their size difference due to electrophoresis and the repeat sequences within the alleles are distinguished from one another by different staining techniques. All sixteen loci are amplified simultaneously.

5.2 Allelic ladder:

The allelic ladder is a collection of different alleles frequently occurred in a given population, which are directly added to the amplified DNA sample and with the internal size standard before electrophoresis .The principal behind the use of allelic ladder is that during the electrophoresis separation the sample fragments separate along with the allelic ladders and the allelic ladders are differentiated from the sample DNA fragments by the software with their fluorescent tag, this makes it convenient to designate which alleles are amplified.

6. CAPILLARY ELECTROPHORESIS:

Electrophoresis designed especially for STR analysis under denatured condition is the capillary electrophoresis. It uses dimethyl polyacrylamide as the matrix and high voltage making the separation rapid. The capillary electrophoresis used for this module was the ABI PRISM 310 GENETIC ANALYZER which has a laser excitation source, a fluorescence detector and auto sampler that holds sample. Sample injection, electrophoresis and data collection are all automated and controlled by a computer. The sizes of the DNA fragments can be calculated by including an internal size standard.

7. PCR Artefacts:

7.1 Stutter peaks:

Stutter peaks appear before or after true alleles and typically have heights less than 15% of the adjacent true allele otherwise called as parent allele. Stutter may mask minor contributors. The stutter peaks are mostly 1-3 Bp less in length than the parent allele. The reason for the occurrence stutter products might be due to the slippage of DNA polymerase during the replication process. When stutter peaks are accidently called as alleles by the software, the profile is interpreted as a mixture profile.

7.2 Micro-variants:

Micro-variants are minute differences in the STR which are regarded to be rare. This could occur probably due to mutations in the primer binding site thereby no amplification of an allele would occur

7.3 Mixture:

A mixture is identified by observing more than two alleles in any locus. Noise and stutter must be disregarded to identify a true mixture. The software helps in differentiation of an allele with the stutter peak. Mixtures can be identified easily with the presence of two or more alleles in a given locus, but sometimes with issues like allelic dropout, heterozygote imbalance or masking of alleles, makes the interpretation and allele designation very complicated and intricate.

7.3 Spikes / Pull ups:

A large peak in a colour which pulls up peak of another colour consistently in the same region , example: a blue peak pulls up a green peak which in turn pulls up the yellow peak . Spikes may occur due to the excess dye , dust particles or air bubbles in the capillary tube.

7.5 Off ladder allele:

Sometimes all the alleles occurring rarely in a population may not be present in the allelic ladder, such alleles are called not called by the software hence known as Off ladder alleles. The same principle applies when the allele falls outside the prescribed allelic regions.

7.6 Issues with PCR inhibition :

The samples obtained from a crime scene are always not pure. It is often found mixed with some other body fluids or with other substances like plant materials, textile dyes, leather, or many be chemical substances; PCR inhibition could be because there is mixture of one or more of these substances with the DNA sample. The inhibition may hinder the DNA amplification, cell lysis and even the addition of nucleotides because of which the alleles are not amplified as it was supposed to be. The profile appears like a profile of that of a degraded DNA which makes the interpretation difficult.

8. Q-PCR results :

“Home Brew” Plexor

Method used Quantity of DNA Method used Quantity of DNA

(ng/ul) (ng/ul)

Chelex saliva 0 Chelex saliva 2.03

Chelex blood 0.08983 Chelex blood 0.231

Qiagen saliva 0.0608 Qiagen saliva 5.02

Qiagen blood 0 Qiagen blood 0.102

8.1 Discussion of the results:

Other DNA extraction methods were not used for comparison because only Chelex and Qiagen methods were used to obtain a DNA profile; hence forth comparison of the efficiency of other methods cannot be established.

Regardless of the sample source and the method of DNA extraction protocol, the Plexor worked well when compared with the Home brew method. This may be because the multi-mix contains all the size standard, appropriate controls, the primers and dyes, which minimized contamination and pipetting errors. Plexor is a commercially available kit, the reagents and the primers used were validated, which makes it perform better. The home brew method more likely may not have appropriate primers and so not validated as in the kits.

With the Plexor multiplexing system, detecting both human and male DNA in every sample will help you make critical decisions about whether to use an autosomal Y STR system without wasting critical sample by performing multiple quantification analysis.

The quantity of DNA also depends on the quantity of the sample retrieved, and also its source. It is surprising that the quantity of DNA taken from blood had less amount of DNA when compared with that of saliva. This probably would be due to the difference in the amount of sample collected from the donor.

The other reasons might be due to the presence of any inhibitors in the reaction mixture which might affect the results; for blood the naturally occurring inhibitor is heme. The buccal swabs are rich in DNA, and do not contain naturally occurring inhibitors but may contain bacteria. Apart from cheek cells, the saliva also contains DNA thus making the source of DNA richer when compared with blood.

The reason why Qiagen did not perform well when compared with Chelex may be due to reason that DNA remained bound to the silica membrane and did not elute during the washing, thereby making only less quantity of DNA available for the PCR reaction.

8.1.1 Sample 82 profile:

The sample 82 is obtained by the Chelex saliva extraction protocol and is over amplified. Over amplification may be due to the presence of excess DNA than the required amount. The PCR reaction is sensitive, and hence even slight excess DNA sample would lead to over amplification. Due to the over amplification many off ladder alleles were observed and Due to this, the profile would often be misinterpreted as a mixed profile. Even small stutter peaks were amplified and were called as alleles by the software. The alleles in the locus 10 and 13 were less amplified when compared with the alleles of other loci. The presence shutter values and off ladder alleles did not actually hinder the inference of the profile because the alleles were certainly over amplified than the stutter peaks and the off ladder alleles. The off ladder alleles were present in almost every locus; this could certainly be due to over amplification.

8.1.2 Sample 83 profile:

The sample 83 corresponds to the profile obtained from a blood swab and the Chelex extraction was followed from DNA extraction from blood.

A good quality profile except for the allele D5S818 was obtained. The amount of DNA added to the PCR amplification mixture was 3.3 ul with that addition of 1 ng/ul to the PCR mixture is achieved. The profile did not contain any prominent spikes except for few stutter peaks occurring just before the peaks of the allele. The alleles for the loci D5 was not called because the allele amplification was much lesser when compared with the amplification of other alleles. If the threshold value was decreased to include the minor allelic peaks, then many other stutter peaks would be called as alleles. For the locus D8 the amplifications was far lesser ,and also for the FGA loci, it seems to be tri allelic due to the shutter values greater than the threshold , but the allele 25 having the maximum height ,is only called omitting 20 and 23 alleles. One off ladder was observed for the locus D18 but did not hinder the interpretation of the profile. For the CSF loci the only one allele 10 was called the allele 13 was not called by the software this may due to poor amplification of that allele.

8.1.3 .Sample 83:

The sample 83 corresponds to the profile obtained from the buccal swab and Qiagen extraction protocol was used to extract DNA. The profile had no allelic peaks except for many peaks which were characteristic of noise and shutter peaks. The reason for not getting a profile could not ascertained, because this just contradicts the amount of DNA obtained from the Q-PCR results where the amount of DNA present was more than required. The reason for not getting a profile may be due to pipetting errors during the addition of DNA sample to the PCR reaction mixture.

8.1.3 .Sample 85:

The sample 85 corresponds to the profile obtained from the Blood swab and QIAamp extraction protocol was used for the DNA extraction. The sample 82 also yielded a complete profile except for the fact that it was over amplified. This may due to addition of excess DNA than the required amount, Since PCR is very sensitive, and it resulted in over amplification of the DNA sample. Artefacts such as shutter peaks and off ladder alleles were observed due to over amplification. Even minute peaks which could have resulted due to small technical defects were even pulled up due t over amplification, because the threshold maintained was just same as for the other samples.

9. Conclusions:

The profiles for the sample 82 and 83 are not included for comparison because one is over amplified which does not yield much of accurate information with presence of many artefacts and for the sample 83, a DNA profile was not obtained.

The samples 83 and 85 are compared to see which extraction protocol yielded good quality profile and how the Q-PCR results determine the quality of the profile. The profile results shows that sample 83 had minimal artefacts, while sample 85 had many PCR related artefacts.

–

Though the sample quantity obtained from the Q-PCR was lesser when compared with the other Chelex sample, it worked better giving almost a complete full and good quality profile with fewer artefacts

The extraction protocol for sample 83 was Chelex and Qiagen for sample 85. Both the methods Chelex and Qiagen work efficiently though both have their own limitations. Bearing the limitations of the protocols and also other various factors, the DNA profiling interpretation is done accordingly.

Literature Review:

1. A. Barbaro, N. S., P. Cormaci, L. Saravo (2003). “DNA profiling by different extraction methods ” International Congress Series 1261: 562-563.

This study aimed to compare the efficiency of different DNA extraction using commercial kits and the ability of each method to yield reliable DNA profiles .DNA samples from liquid blood, old blood stains, cigarette buds, semen stains and hairs.

The QIAamp lit worked so well with an excellent extraction method and purification with traces of inhibitors, though loss in DNA occurred around 30%.

While even the Chelex extraction method seems to work well with low cost therefore its commonly used for samples from cigarette buds, semen , blood stains, perhaps there is no separate step unlike the Qiagen to remove the inhibitors,. “Finally the paper concludes saying that the right choice of the DNA extraction method and an accurate DNA quantification are very important step in the analytical procedure to ensure optimal results “.

2. V. Castella , N. D.-S., C. Brandt-Casadevall, P. Mangin (2006). “Forensic evaluation of the

QIAshredder/QIAamp DNA extraction procedure ” Forensic Science International 156: 70-73.

This paper aims to evaluate the efficiency of QIAamp the commercially available kit by comparing the efficiency of QIAamp with Chelex and Phenol – Chloroform with various DNA samples from blood, saliva, and cotton swabs, and cigarette buds etc. Only 61% of the samples yielded conclusive results Chelex and Phenol Chloroform methods were used, but for QIAamp 82% of the samples were successful. This paper also suggests the use of QIAshredder column in conjunction with the QIAamp system to further improve the recovery of DNA from blood and saliva.” Overall, the use of QIAamp was more successful than the Chelex and Phenol-Chloroform methods and thus states that the QIAamp is efficient when the samples contained smaller amounts of DNA, and also for samples which contained inhibitors. (Luke Forster 2008)