Polymerase Chain Reaction: Basic Principles and Routine Practice

Kolmodin, Lori A.; Williams, J F

doi:10.1385/1-59259-038-1:569

Cited by 10 publications

(11 citation statements)

References 24 publications

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“…The increase in the starting volume of the DNA template did not result in satisfactory amplification, most probably due to accompanying excess in the EDTA‐containing elution buffer residue, which has a potential to inhibit the PCR reaction by chelation of magensium ions . In theory, even a single molecule of DNA could be successfully amplified, and the amount of genomic DNA appropriate for PCR has been determined to up to 1 μg . Yet, here we dealt with a difficult DNA template, thus, good concentration DNA of at least 2 μg/ml proved to be a baseline condition for successful amplification.…”

Section: Discussionmentioning

confidence: 96%

Optimization of PCR Conditions for Amplification of GC-RichEGFRPromoter Sequence

Obradovic

Jurišić

Tos̆ić

et al. 2013

J. Clin. Lab. Anal.

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Section: Discussionmentioning

confidence: 96%

Optimization of PCR Conditions for Amplification of GC-RichEGFRPromoter Sequence

Obradovic

Jurišić

Tos̆ić

et al. 2013

J. Clin. Lab. Anal.

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“…Optimal concentration of 10% was determined after testing three different options, which was within the recommended range of 1-10% (Grunenwald, 2003;Kolmodin and Birch, 2002).…”

Section: Discussionmentioning

confidence: 99%

PCR Amplification Protocol for GC Rich Protamine Gene from Chicken Testis cDNA

Sharma¹

2015

Adv. Anim. Vet. Sci.

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| Amplification of GC rich templates using PCR is usually difficult compared to non-GCrich targets. GC rich regions produce complex inter and intra strand folding (hairpins and loops) due to higher hydrogen bonding with neighbouring cytosine and guanine. These secondary structures in DNA are resistant to melting and cause Taq DNA polymerases to stall; they also hamper primer annealing, resulting in incomplete or non-specific amplifications. The aim of this study was to develop an easy PCR protocol to amplify very high GC rich (88% in the coding region) protamine gene of chicken. The cDNA used for amplification was synthesised from testis RNA and PCR products were detected by agarose gel electrophoresis. Protamine amplicon successfully amplified only with Go Taq DNA polymerase in the presence of DMSO. Optimization of MgCl 2 concentration, buffer strength, annealing temperature, DMSO and the DNA template concentration, were important in the PCR reaction resulting in successful amplification. In addition, PCR started directly at hot plate is also important to get rid of primer-dimer formation.

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“…При цьо му най час ті ше ви ко ристо ву ють мі то хон д рі аль ні гени, на прик лад ген ци то хро мок си да зи чи ген ри бо сом ної РНК або хро мо сом ний ген ри бо сом ної РНК (Hebert et al, 2003;Kress et al, 2005;Epp et al, 2012). У зраз ках ґрун ту, оса ду чи води кіль кість eDNA зви чай но не знач на, тому її не об хід но кло ну ва ти до об ся гу, не об хід но го для по даль шо го ана лі зу, за до по мо гою так зва ної по лі ме раз ної лан цю го вої ре ак ції (polymerase chain reaction, PCR) (Kolmodin et al, 2002;Garibyan et al, 2013). Ос кіль ки тех но ло гія ДНКмар ке рів вия ви ла ся вель ми ко рис ною для мо ні то рин гу давньо го і су час но го біо різ но ма ніт тя різ них еко систем, на ра зі від бу ва єть ся пе ре хід від од но мар кер но го ана лі зу ви дів та уг ру по вань ор га гри ба ми є дріж джі (од но клі тин ні аско та ба зи діо мі це ти), які роз ви ва ють ся у за бруд не ній воді, планк то ні, на мак ро во до рос тях тощо (Kohlmeyer et al, 1979).…”

Section: гри би в еко систе мах су хо до луunclassified

Environmental DNA as a tool for ecological monitoring of fungal communities

Pomohaybo¹,

Makarenko²

2017

Ukr. Bot. J.

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An overview of recently published data on fungal communities based on the environmental DNA technology is provided. In most cases, these scarce data result from the wide range biodiversity studies of eukaryotes while detecting species richness of fungi from eDNA is still poorly studied. However, recent eDNA analyses have already revealed numerous undescribed taxa of fungi in various ecosystems. They also demonstrated that eDNA technology may considerably increase the total number of fungal species comparatively with those described so far using traditional methods. Environmental DNA barcoding as an efficient technique for detecting fungal diversity in various ecosystems provides new insights into the evolution of fungi.

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Polymerase Chain Reaction: Basic Principles and Routine Practice

Cited by 10 publications

References 24 publications

Optimization of PCR Conditions for Amplification of GC-RichEGFRPromoter Sequence

Optimization of PCR Conditions for Amplification of GC-RichEGFRPromoter Sequence

PCR Amplification Protocol for GC Rich Protamine Gene from Chicken Testis cDNA

Environmental DNA as a tool for ecological monitoring of fungal communities

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