PCR Amplification of Clock and Adcyap1 genes with EmeraldAmp® GT PCR Master Mix in Avian species for polymorphism elucidation

Louis-Stéphane Le Clercq, Desiré Lee Dalton, Antoinette Kotzé, Paul Grobler

Published: 2023-11-24 DOI: 10.17504/protocols.io.6qpvrdwk3gmk/v1

Abstract

This PCR protocol is used to amplify Clock and Adcyap1 gene regions in avian species which have previously shown polymorphisms, such as poly-Q runs, that correlated to migration phenology. It was tested and optimized in Woodlands kingfisher (Halcyon senegalensis) and Diederik cuckoos (Chrysococcyx caprius). The primers were designed based on those previously used by Johnson et al. (2007) and Steinmeyer et al. (2009) by comparing the relevant gene sequences for chickens (Galus galus) with several other available avian species to select primers that would account for the most common variations in primer regions, enabling more universal amplification. Individual clock gene sequences were retrieved from Genbank and aligned in BioEdit 7.2. Primers were then selected based on the annotated regions. The assay was designed using 25 μL (half) reactions of EmeraldAmp® GT PCR Master Mix, which is premixed with loading buffer for easy gel loading following PCR and does not require a long initial denaturation step (thereby shortening the run time). Gel electrophoresis was able to confirm successful amplification of a product ±280 bp long in both species. The same primers were subsequently used for sanger sequencing. A BLAST search of the resulting sequences confirmed the identity of the amplified regions.

Before start

Thaw reagents On ice .* Wipe workspace with 10% volume Bleach, followed by 70% volume Ethanol, and ddH₂O before (and after).

UV the relevant laminar flow cabinets.

Attachments

Clock_anot_Primers+Product [BioEdit].bio Adcyap anot Primers+Product [BioEdit].bio NZG_LeClercq_PCR_template Clock and Adcyap1 genes.xlsx

Steps

Master Mix set-up

Prepare Master Mix and Samples* for PCR.

*Sample information has been deposited to BioSample and associated to the BioProject (PRJNA737185) which used this protocol.

(An experiment template is included in excel format.)

1.1.

Set up the following Master Mix with for a 25µL reaction in a DNA-free lab and laminar flow cabinet.

A	B	C	D
EmeraldAmp® GT PCR Master Mix	X2	X1	12.5
Forward primer	10 µM	0.2 µM	0.5
Reverse primer	10 µM	0.2 µM	0.5
Nuclease free water	-	-	9.5

Summary of components to add to Master Mix with the original and final concentrations as well as the relative volume in μL

Working solutions of forward (Clk F1) and reverse (Clk R1) primers.

1.2.

Add 23µL to 2µL* in individual thin-walled PCR tubes in a DNA-loading laminar flow cabinet.

PCR reactions prepared to run thermal cycles.

*DNA isolated from avian blood samples may be highly concentrated, ensure that it is still less that 500ng of DNA per reaction.

Thermal cycling

Program and run the following thermal cycling profile on a thermal cycler, e.g.

Equipment

Value	Label
SimpliAmp Thermal Cycler	NAME
PCR	TYPE
Applied Biosystems	BRAND
A24811	SKU
Any standard PCR thermocycler will suffice	SPECIFICATIONS

40Cycles of:

Denaturation at 98°C for 0h 0m 10s
Annealing at 60°C for 0h 0m 30s
Elongation at 72°C for 0h 1m 0s

Infinite hold at 4°C until ready for next steps.

Example of PCR run lasting approximately 1h30.

Electrophoresis

Confirm success of amplification by TAE/TBE electrophoresis.

3.1.

Prepare a 2% (v/v) gel with and , pre-stained with using a casting tray and comb with sufficient wells.

Resolution capacity of different concentrations of gels .

A	B
0.3	5 to 60 kbp
0.6	1 to 20 kbp
0.7	0.8 to 10 kbp
0.9	0.5 to 7 kbp
1.2	0.4 to 6 kbp
1.5	0.2 to 3 kbp
2.0	0.1 to 2 kbp

Concentration (%) of agarose gels and their efficient range of separation in kilo base pairs.

Amount of agarose required for a small (50 mL) and large (100 mL) gel.

A	B	C
0.3	150mg	300mg
0.6	300mg	600mg
0.7	350mg	700mg
0.9	450mg	900mg
1.2	600mg	1.2g
1.5	750mg	1.5g
2.0	1g	2g

Concentration (%) of gels and their required amount of agarose.

*Note: is usually added at a concentration of 1µL per 10mL

3.2.

Load 4µLof PCR product to the gel alongside a molecular weight marker, e.g. , and run at 60Volt for 0h 30m 0s . Possible settings for the

Equipment

Value	Label
PowerPac Basic Power Supply	NAME
Power Supply	TYPE
Bio-Rad Scientific	BRAND
1645050	SKU

are:

A	B
< 1kbp	5 V/cm
1-12 kbp	4-10 V/cm
>12 kbp	1-2 V/cm

Ideal voltages for resolving different size fragments.

3.3.

Visualize and capture gel on an appropriate imager and paired software, e.g.

Equipment

Value	Label
Gel Doc XR+ Gel Documentation System	NAME
Gel Documentation System	TYPE
Bio-rad Laboratories	BRAND
1708195	SKU

Citation

Gel image of molecular marker and clock gene amplicons for Diederik cuckoo:

Positive amplification of clock genes viewed on a 1.5% TAE-Agarose gel.

Amplicon purification

Purify the positive amplicons with prior to sequencing.

4.1.

Mix 5µL with 2µL for a total volume of 7µL .

4.2.

Incubate at 37°C for 0h 15m 0s to degrade PCR primers and short products.

4.3.

Incubate at 80°C for 0h 15m 0s to inactivate the .

Exo-SAP digestion and inactivation cycles.

4.4.

The PCR product is now ready for use in DNA sequencing*, SNP analyses, or other primer-extension applications.

*See Clock genes sequencing protocol (https://protocols.io/view/abi-sanger-sequencing-of-avian-clock-genes-to-eluc-bvydn7s6)

PCR Amplification of Clock and Adcyap1 genes with EmeraldAmp® GT PCR Master Mix in Avian species for polymorphism elucidation

Abstract

Before start

Attachments

Steps

Master Mix set-up

Thermal cycling

Electrophoresis

Amplicon purification

推荐阅读