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Alu SINE analyses of 3,000-year-old human skeletal remains: a pilot study.

Kothe M, Seidenberg V, Hummel S, Piskurek O - Mob DNA (2016)

Bottom Line: For 26 of the 30 Alu loci investigated, definite results were obtained.The third individual revealed less well-preserved DNA, which results in allelic dropout or complete amplification failures.Human population genetic studies and other large-scale investigations would provide insight into Alu SINE-based microevolutionary processes in humans during the last few thousand years and help us comprehend the evolutionary dynamics of our genome.

View Article: PubMed Central - PubMed

Affiliation: Department of Historical Anthropology and Human Ecology, Georg-August-University, 37073 Göttingen, Germany.

ABSTRACT

Background: As Short Interspersed Elements (SINEs), human-specific Alu elements can be used for population genetic studies. Very recent inserts are polymorphic within and between human populations. In a sample of 30 elements originating from three different Alu subfamilies, we investigated whether they are preserved in prehistorical skeletal human remains from the Bronze Age Lichtenstein cave in Lower Saxony, Germany. In the present study, we examined a prehistoric triad of father, mother and daughter.

Results: For 26 of the 30 Alu loci investigated, definite results were obtained. We were able to demonstrate that presence/absence analyses of Alu elements can be conducted on individuals who lived 3,000 years ago. The preservation of the ancient DNA (aDNA) is good enough in two out of three ancient individuals to routinely allow the amplification of 500 bp fragments. The third individual revealed less well-preserved DNA, which results in allelic dropout or complete amplification failures. We here present an alternative molecular approach to deal with these degradation phenomena by using internal Alu subfamily specific primers producing short fragments of approximately 150 bp.

Conclusions: Our data clearly show the possibility of presence/absence analyses of Alu elements in individuals from the Lichtenstein cave. Thus, we demonstrate that our method is reliably applicable for aDNA samples with good or moderate DNA preservation. This method will be very useful for further investigations with more Alu loci and larger datasets. Human population genetic studies and other large-scale investigations would provide insight into Alu SINE-based microevolutionary processes in humans during the last few thousand years and help us comprehend the evolutionary dynamics of our genome.

No MeSH data available.


Related in: MedlinePlus

The photo shows seven successfully amplified amplicons of an internal Alu primer-based amplification. The expected fragment lengths vary from 118-194 bp. The marks on the base pair ladder are situated at 150 bp and 350 bp. For these seven Alu loci, the presence band for DO 3750 was proved via internal Alu amplification. The asterisks indicate reverse Alu inserts. In these cases, the primer pairings are an internal Alu primer with the reverse Alu flanking primer, whereas the samples without asterisk were amplified with an internal Alu primer and the forward Alu flanking primer
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Fig2: The photo shows seven successfully amplified amplicons of an internal Alu primer-based amplification. The expected fragment lengths vary from 118-194 bp. The marks on the base pair ladder are situated at 150 bp and 350 bp. For these seven Alu loci, the presence band for DO 3750 was proved via internal Alu amplification. The asterisks indicate reverse Alu inserts. In these cases, the primer pairings are an internal Alu primer with the reverse Alu flanking primer, whereas the samples without asterisk were amplified with an internal Alu primer and the forward Alu flanking primer

Mentions: Obviously incomplete and incongruent results were subjected to an alternative molecular approach. Using an internal Alu primer, the predicted fragment length of the amplicon was reduced to ~150 bp (Fig. 1). The internal primers were designed based on an alignment of Alu sequences of the respective subfamily and consequently are very specific to each AluY subfamily as described by Nelson et al. [49] or Kass and Batzer [50]. This type of amplification worked in seven cases for the sample DO 3750 (Fig. 2). The heterozygous results for Alu_16, Alu_26 and Alu_27 for the daughter (‘CR’ in Table 1) represent a combination of both amplification approaches. Further internal Alu primer analyses were not possible, due to a depleted DNA extract (Alu_4, Alu_25; marked red). Loci with exclusively absence bands for the prehistoric individuals, in particular, should be checked by internal Alu amplification. The advantage of this method is that the amplification of short fragments (usually ~150 bp) still proves the presence of an insert. In this study, this approach was applied only in those cases where the Alu amplification results are not in concordance with the family situation or where the amplification totally failed for DO 3750. Based on previous analyses on this prehistoric triad, it is known that the DNA is less well-preserved in DO 3750 and best-preserved in DO 1911. Consequently, the chance of allelic dropout events for DO 3750 is more likely than for DO 3756 and DO 1911. Fragments of such short lengths (~150 bp) are usually not affected by allelic dropout. However, the internal primer approach cannot be applied in isolation because it does not indicate heterozygous states.Fig. 1


Alu SINE analyses of 3,000-year-old human skeletal remains: a pilot study.

Kothe M, Seidenberg V, Hummel S, Piskurek O - Mob DNA (2016)

The photo shows seven successfully amplified amplicons of an internal Alu primer-based amplification. The expected fragment lengths vary from 118-194 bp. The marks on the base pair ladder are situated at 150 bp and 350 bp. For these seven Alu loci, the presence band for DO 3750 was proved via internal Alu amplification. The asterisks indicate reverse Alu inserts. In these cases, the primer pairings are an internal Alu primer with the reverse Alu flanking primer, whereas the samples without asterisk were amplified with an internal Alu primer and the forward Alu flanking primer
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4836192&req=5

Fig2: The photo shows seven successfully amplified amplicons of an internal Alu primer-based amplification. The expected fragment lengths vary from 118-194 bp. The marks on the base pair ladder are situated at 150 bp and 350 bp. For these seven Alu loci, the presence band for DO 3750 was proved via internal Alu amplification. The asterisks indicate reverse Alu inserts. In these cases, the primer pairings are an internal Alu primer with the reverse Alu flanking primer, whereas the samples without asterisk were amplified with an internal Alu primer and the forward Alu flanking primer
Mentions: Obviously incomplete and incongruent results were subjected to an alternative molecular approach. Using an internal Alu primer, the predicted fragment length of the amplicon was reduced to ~150 bp (Fig. 1). The internal primers were designed based on an alignment of Alu sequences of the respective subfamily and consequently are very specific to each AluY subfamily as described by Nelson et al. [49] or Kass and Batzer [50]. This type of amplification worked in seven cases for the sample DO 3750 (Fig. 2). The heterozygous results for Alu_16, Alu_26 and Alu_27 for the daughter (‘CR’ in Table 1) represent a combination of both amplification approaches. Further internal Alu primer analyses were not possible, due to a depleted DNA extract (Alu_4, Alu_25; marked red). Loci with exclusively absence bands for the prehistoric individuals, in particular, should be checked by internal Alu amplification. The advantage of this method is that the amplification of short fragments (usually ~150 bp) still proves the presence of an insert. In this study, this approach was applied only in those cases where the Alu amplification results are not in concordance with the family situation or where the amplification totally failed for DO 3750. Based on previous analyses on this prehistoric triad, it is known that the DNA is less well-preserved in DO 3750 and best-preserved in DO 1911. Consequently, the chance of allelic dropout events for DO 3750 is more likely than for DO 3756 and DO 1911. Fragments of such short lengths (~150 bp) are usually not affected by allelic dropout. However, the internal primer approach cannot be applied in isolation because it does not indicate heterozygous states.Fig. 1

Bottom Line: For 26 of the 30 Alu loci investigated, definite results were obtained.The third individual revealed less well-preserved DNA, which results in allelic dropout or complete amplification failures.Human population genetic studies and other large-scale investigations would provide insight into Alu SINE-based microevolutionary processes in humans during the last few thousand years and help us comprehend the evolutionary dynamics of our genome.

View Article: PubMed Central - PubMed

Affiliation: Department of Historical Anthropology and Human Ecology, Georg-August-University, 37073 Göttingen, Germany.

ABSTRACT

Background: As Short Interspersed Elements (SINEs), human-specific Alu elements can be used for population genetic studies. Very recent inserts are polymorphic within and between human populations. In a sample of 30 elements originating from three different Alu subfamilies, we investigated whether they are preserved in prehistorical skeletal human remains from the Bronze Age Lichtenstein cave in Lower Saxony, Germany. In the present study, we examined a prehistoric triad of father, mother and daughter.

Results: For 26 of the 30 Alu loci investigated, definite results were obtained. We were able to demonstrate that presence/absence analyses of Alu elements can be conducted on individuals who lived 3,000 years ago. The preservation of the ancient DNA (aDNA) is good enough in two out of three ancient individuals to routinely allow the amplification of 500 bp fragments. The third individual revealed less well-preserved DNA, which results in allelic dropout or complete amplification failures. We here present an alternative molecular approach to deal with these degradation phenomena by using internal Alu subfamily specific primers producing short fragments of approximately 150 bp.

Conclusions: Our data clearly show the possibility of presence/absence analyses of Alu elements in individuals from the Lichtenstein cave. Thus, we demonstrate that our method is reliably applicable for aDNA samples with good or moderate DNA preservation. This method will be very useful for further investigations with more Alu loci and larger datasets. Human population genetic studies and other large-scale investigations would provide insight into Alu SINE-based microevolutionary processes in humans during the last few thousand years and help us comprehend the evolutionary dynamics of our genome.

No MeSH data available.


Related in: MedlinePlus