What Primer Do You Use to Amplif Dna for Mtdna Contol Region
Mitochondrial Dna B Resour. 2017; 2(1): 99–100.
Distension of mtDNA control region in opportunistically collected bird samples belonging to nine families of the order Passeriformes
Ashutosh Singh
aDepartment of Brute Environmental and Conservation Biology, Wildlife Institute of India, Dehradun, Republic of india;
bDepartment of Environmental Scientific discipline and Zoology, Gurukula Kangri Academy, Haridwar, India;
Ajit Kumar
aDepartment of Animal Ecology and Conservation Biology, Wildlife Institute of Bharat, Dehradun, Republic of india;
bDepartment of Environmental Science and Zoology, Gurukula Kangri University, Haridwar, Bharat;
Ramani Suresh Kumar
cDepartment of Endangered Species Direction, Wildlife Institute of Republic of india, Dehradun, India
Dinesh Bhatt
bSection of Environmental Science and Zoology, Gurukula Kangri University, Haridwar, India;
Sandeep Kumar Gupta
aDepartment of Fauna Ecology and Conservation Biological science, Wildlife Institute of India, Dehradun, India;
Abstract
We describe six sets of primers for amplifying the mitochondrial command region (CR) of various bird species belonging to nine families of the lodge Passeriformes. These overlapping primers, with both short and long fragments, yielded an approximately 1 kb fragment of the CR. The short length of the amplified production makes the primers suitable for degraded DNA samples. These primers were used on a broad range of bird species for amplifying and sequencing the highly variable portion of the CR. The primers proved to exist a valuable tool for studying the population genetics of bird species. The dissimilar sets of primers provide the researcher a pick of markers for different sample types and studies.
Keywords: Mitochondrial control region, birds, not-invasive samples, PCR
Introduction
Molecular genetics is existence used increasingly in various conservation applications. It is useful in determining the level of genetic variation, phylogenetics and phylogeography with a high level of accurateness. Due to the presence of conserved sites in mitochondrial Deoxyribonucleic acid (mtDNA) regions such equally the 12s, 16s and cytochrome b gene, these fragments are widely used for species identification (Kocher et al. 1989; Wan et al. 2004). The mtDNA control region is helpful in identifying meaning conservation units for those species that are historically isolated (Wenink et al. 1994; Gupta et al. 2015). Besides, knowledge nigh the variability of the hypervariable control region is helpful in identifying lower-category taxa such as species or sub-species. The mitochondrial genome is highly variable in avian species (Wenink et al. 1994). The control region (CR) often evolves faster than the rest of the mitochondrial genome (Baker & Marshall 1997). This variability of the CR has made information technology a powerful tool for studying the genetic structures of populations. Because of the high variability in the CR and the to the lowest degree conserved sites, designing conserved primers is often a challenging job (Arif & Khan 2009). Moreover, bird genetics largely involves not-invasively collected biological samples (shed feathers, faeces, shells of cleaved/hatched eggs). Amplifying long fragments of the CR from such opportunistically collected samples is a challenging chore. A set up of primers amplifying shorter fragments will be useful for such samples (Gupta et al. 2014). In this work, we describe a panel of primers for amplification of the mtDNA CR of selected bird species from degraded DNA.
Materials and methods
Primer design
The complete mitochondrial genomes of 28 bird species belonging to 9 families (Muscapidae, Polioptilidae, Emberizidae, Estrildidae, Viduidae, Nectariniidae, Passeridae, Prunellidae, Fringillidae) were obtained from GenBank and aligned using Clustal W multiple alignments (Thompson et al. 1994). On the basis of sequence similarity, nosotros designed four forward and 4 reverse primers targeting the CR (Table 1).
Tabular array 1.
Primer sequences for the distension of control region of bird species and expected length of their amplicons.
| Due south. No. | Primer proper noun/combination | Sequence (5′-3′)/Expected amplicon length |
|---|---|---|
| 1 | FLCF1 | GAA TGG GGT CAA AGT GCA TCA GT |
| two | FLCF2 | TGA TGG ACA TGT CAA GAG GAA G |
| 3 | FLCF3 | GG CGC AAA AGA GCA AGT |
| 4 | FLCR1 | Deed TGC TCT TTT GCG CC |
| 5 | FLCF4 | GTA GCT CGG TTC TCG TGA GAA |
| half-dozen | FLCR2 | TTC TCA CGA GAA CCG AGC TAC |
| viii | FLCR3 | CCT GAA AAG CCG CTG TTA T |
| nine | FLCR4 | TCC ATC TCC AGC TCC CAA AGC |
| Primer combination and expected length | ||
| i. | FLCF1+ FLCR1 | 320 (bp) |
| ii. | FLCF1 + FLCR2 | 410 (bp) |
| three. | FLCF2+ FLCR1 | 217 (bp) |
| four. | FLCF2+ FLCR2 | 300 (bp) |
| v. | FLCF4+ FLCR3 | 510 (bp) |
| vi. | FLCF3+ FLCR4 | 569 (bp) |
Sample collection, Deoxyribonucleic acid extraction and distension
We collected shed feathers, cleaved egg shells and blood samples of 28 different bird species through a field survey. Genomic Deoxyribonucleic acid was extracted using the standard phenol–chloroform method (Sambrook et al. 1989) and subjected to PCR distension using the primer combination described in Table one. The amplification was carried out in a 20 μl reaction volume containing 1 μl of the extracted DNA, 100 μM of dNTPs, 4 pmol of each primer, ane.five mM MgCl2, 0.5 units of AmpliTaq Gilded (Life Technologies) and one × PCR buffer (10 mM Tris–HCl, pH 8.3, and 50 mM KCl). The PCR conditions were the post-obit: initial denaturation at 95 °C for ten min, followed by 35 cycles of denaturation at 95 °C for 45 southward, annealing at 56 °C for 45 southward and extension at 72 °C for ninety s. The final extension was at 72 °C for ten min. The PCR products were electrophoresed on 2% agarose gel, stained with ethidium bromide (0.v mg/ml) and visualized under a UV transilluminator. The PCR products obtained were sequenced directly in 3130 Genetic Analyzer (Applied Biosystems) from both directions.
Results and conclusion
DNA from 28 different bird species was successfully amplified. The primers described in this report were useful in generating the DNA sequence database and were helpful in identifying species and sub-species and in phylogeographic differentiation. The utilise of different lengths of the CR amplicon in single PCR was a useful approach to amplifying the combination of brusk and longer Deoxyribonucleic acid fragments found in degraded samples (Figure 1). The brusque length of the amplified product makes these primers suitable for highly degraded samples. Therefore, the overlapping fragments generated by the primer set were helpful in covering the longer portions of the CR. The PCR conditions described in this article worked consistently for all the primers mentioned. As well, these primers could be used with a large range of bird species. Hence, they are tin can be a valuable tool for studying population genetics and identifying evolutionarily meaning units (ESUs).
Gel prototype showing the amplification event of mtDNA command region from the DNA extracted from bird samples using primer FLCF1 + FLCR1 (lane ane); FLCF1 + FLCR2 (lane 2); FLCF2 + FLCR1 (lane 3); FLCF2 + FLCR2 (lane iv); FLCF4 + FLCR3 (lane 5); FLCF3 + FLCR4 (lane 6).
Acknowledgements
This study was funded by Scientific and Engineering Enquiry Board (SERB), Department of Science and Technology (DST), Regime of Bharat through grant number SR/So/Equally-85/2012. We thank Dr V. B. Mathur, Director, WII; Dr Grand. S. Rawat, Dean, WII; Dr Pratap Singh, WII; Dr Y. V. Jhala, WII and Dr Dhananjai Mohan, Additional PCCF, Uttarakhand Forest Department for their kind support and Suresh Kumar Rana for help with sample collection. We thank the woods departments of Jammu & Kashmir, Uttarakhand, Sikkim, Due west Bengal and Arunachal Pradesh for according permissions to conduct the study.
Disclosure statement
There is no disharmonize of involvement. All authors accept read and agree with the content of the paper.
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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7800550/
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