May 04, 2026
Human GBA1 Gene PCR and Sanger Sequencing Protocols
- nahid tayebi1,2,3
- 1Medical Genetics Branch;
- 2National Human Genome Research Institute;
- 3National Institutes of Health, Bethesda, MD, USA.
Protocol Citation: nahid tayebi 2026. Human GBA1 Gene PCR and Sanger Sequencing Protocols. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbk34qgpk/v1
Manuscript citation:
Tayebi N, Lichtenberg J, Hertz E, Sidransky E. Is Gauchian genotyping of GBA1 variants reliable?. Commun Biol. 2025;8(1):718. Published 2025 May 9. doi:10.1038/s42003-025-08059-y
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 06, 2025
Last Modified: May 04, 2026
Protocol Integer ID: 219709
Keywords: ASAPCRN, GBA1, Sanger Sequencing, protocol for human gba1 gene pcr, human gba1 gene pcr, gba1, sanger sequencing protocol, sanger sequencing protocols the presence, gbap1, complex recombinant allele, gene, frequency of nonequal pairing, homologous pseudogene, chromosome, variant detection
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000458
Abstract
The presence of a highly homologous GBA1 pseudogene, GBAP1, located approximately 16 kb downstream from the gene, complicates variant detection and sequence analyses, as highly homologous pseudogenes increase the frequency of nonequal pairing of chromosomes, resulting in complex recombinant alleles. Here we provide a protocol for Human GBA1 Gene PCR and Sanger Sequencing
Procedure
Isolate high-molecular-weight DNA from patients' and controls' blood or cultured cell lines.
Amplify all exonic and most intronic regions of the GBA1 gene, not the pseudogene, in three fragments by PCR, using the following conditions:
Exon 1-5 using primers E1F and 39R, the PCR annealing temperature is 57°C, and the product size is 2,960 bp. The primer sequences: forward primer (E1F), 5’-CCTAAAGTTGTCACCCATAC-3’, and the reverse primer (39R), 5’-AGCAGACCTACCCTACAGTTT-3’.
Exon 5-8 using 127F and 66R primers. The annealing temperature is 60°C, and the product size is 3,163 bp. Forward primer (127F): 5’-GACCTCAAATGATATACCTG-3’ and the reverse primer (66R) 5’-AGTTTGGGAGCCAGTCATTT-3’.
Exon 8-11 using cor8F and 172R primers. The annealing temperature is 61°C and the product size is 1,750 bp. Forward primer (cor8F) 5’-TGTGTGCAAGGTCCAGGATCAG-3’ and the reverse primer (172R) 5’-ACCACCTAGAGGGGAAAGTG-3’.
General PCR Reaction: (ExTaq enzyme, buffer, and dNTP from TakaRa Bio USA, product # TAK RR001A. TakaRa conditions are used for Thermal Cycler.
Purify the three fragments using MinElute PCR Purification Kit, QIAGEN, CA, Cat #: 28004.
For cycle sequencing, using BigDye™ Terminator v3.1 Ready Reaction Mix, BigDye™ Terminator v1.1 & v3.1 5X Sequencing Buffer, purified PCR, 50-150 ng, sequencing primer, and UltraPure™ DNase/RNase-Free Distilled Water, follow the link protocol to run the sequencing reactions.
Add 80-150 ng of DNA to each well.
Add 1 μL of each primer.
Prepare the master mix in excess: For 1 μL of DNA: Reagent Volume (μL) Terminator buffer 2, Water 5, Big dye 1. Reach a total volume of 10 μL. Cap wells, centrifuge briefly, and set up in a thermocycler.
BigDye Xterminator purification:
ThermoFisher purification Kit, Cat: 4376486, and the protocol from Applied Biosystems, following the link protocol.
Agencourt CleanSEQ Dye-Terminator: Agencourt CleanSEQ is a magnetic bead-based sequencing purification system, Beckman Coulter, Cat: A29154, follow the protocol from the provided link.
Protocol for the 96-Well Format:
Shake the CleanSEQ reagent to fully resuspend the magnetic beads before using. The reagent should appear homogeneous and consistent in color. Ensure that no visible bead pellet remains in the bottle.
Add 10 μL of CleanSEQ reagent to each sample; use 10 μL of CleanSEQ regardless of the sequencing reaction volume.
Add 85% ethanol to each sample according to the following formula: Volume of 85% Ethanol = 2.077 × (10 μL + Sample Volume). Pipette mix seven times, or until the solution is homogeneous throughout each well (ethanol floats to the top of the sample, while the CleanSEQ sinks to the bottom). IMPORTANT: Mix the layers thoroughly to ensure complete binding of the sequencing products to the magnetic beads.
Place the sample plate onto a Beckman Coulter Agencourt SPRIPlate 96R for 3-5 minutes, or any magnetic rack, until the solution is clear. The magnetic beads form a ring or a crescent on the side of the well.
Aspirate the cleared solution (supernatant) from the plate and discard it, ensuring that all liquid is removed from every well. To avoid disturbing the beads, place the pipette tip at the bottom of the well when aspirating. Remove as much supernatant as possible since it contains excess fluorescent dye and contaminants.
Dispense 100 μL of 85% ethanol into each well. Wait at least 30 seconds for the beads to resettle before proceeding to the next step. IMPORTANT: Perform this step while the plate is seated on the magnet. Mixing or resuspending the beads during this step is not necessary.
Completely remove the ethanol and discard. Ensure that all liquid is removed from every well. To avoid disturbing the beads, place the pipette tip at the bottom of the well when aspirating. Remove as much ethanol as possible, since it contains excess fluorescent dye and contaminants.
Repeat steps 6 and 7 for a total of two 85% ethanol washes.
Let the samples dry for 10 minutes at room temperature.
Add 40 μL of water into each well. Incubate the plate for 2–5 minutes at room temperature to elute.
Allow the sample plate to separate on the magnet for 3–5 minutes or until the solution is clear.
Transfer 30 μL of the clear sample into a new plate for loading on the sequencing machine.
Seal the samples and store at 4°C for up to 24 hours before loading. If the samples will not be loaded within 24 hours, store them at –20°C. Samples can be kept at –20°C for approximately one month.