Activity 4-4 - DNA Sequencing with SeqStudio Genetic Analyzer

Introduction to DNA Sequencing with SeqStudio

DNA sequencing is the process of determining the precise order of nucleotides—adenine (A), thymine (T), cytosine (C), and guanine (G)—in a DNA molecule. Understanding this sequence is essential in modern biology, biotechnology, and medicine because it allows researchers to identify genes, mutations, and genetic variations associated with traits or diseases. In this lab, we use a technique called Sanger sequencing, which is a widely used and highly accurate method suitable for sequencing single genes or short DNA fragments (usually up to 1000 base pairs). Sanger sequencing works by copying a single-stranded DNA template while randomly incorporating fluorescently labeled chain-terminating nucleotides (called dideoxynucleotides or ddNTPs). These special nucleotides stop DNA synthesis at random points, resulting in fragments of different lengths. Each ddNTP is tagged with a different fluorescent dye (corresponding to A, T, G, or C), and once the sequencing reaction is complete, the fragments are separated by size using capillary electrophoresis. The fluorescent labels are detected by a laser, allowing the instrument to "read" the DNA sequence based on the color of each terminal base in order from smallest to largest fragment.

The instrument used in this lab is the Applied Biosystems SeqStudio Genetic Analyzer, a compact, automated machine that performs capillary electrophoresis and sequence detection. It uses a small cartridge with four capillaries, which means it can process up to four samples at a time. To run properly, the instrument requires a buffer-filled Cathode Buffer Cartridge (CDC) and carefully prepared samples that have been cleaned of contaminants such as excess primers, salts, and leftover PCR reagents.

Before sequencing, DNA needs to be amplified using Polymerase Chain Reaction (PCR). PCR makes millions of copies of a specific DNA region using primers—short DNA sequences that target the beginning and end of the region you want to amplify. For sequencing, two separate reactions are usually set up: one with a forward primer and one with a reverse primer, so that both strands of the DNA can be read independently. This gives you better sequence coverage and helps confirm accuracy. After PCR, the amplified DNA must be purified. This is crucial because any residual primers or nucleotides can interfere with the sequencing chemistry, resulting in poor-quality reads or background noise. The purification process includes both enzymatic cleanup (using enzymes that degrade leftover primers and dNTPs) and column-based purification (using silica membranes to bind and wash DNA). Once cleaned, the DNA is quantified, often using a Nanodrop spectrophotometer, to ensure there is enough material for sequencing and that it's free of contaminants.

The actual sequencing reaction involves mixing the purified DNA with the BigDye Terminator v3.1 Cycle Sequencing Kit, which contains DNA polymerase, normal dNTPs, fluorescently labeled ddNTPs, and necessary buffers. This reaction is cycled in a thermal cycler, similar to PCR, to produce the fluorescently labeled DNA fragments of varying lengths. Once samples are prepared, they are loaded into a 96-well plate designed for the SeqStudio instrument. The plate is covered with a septa strip to prevent evaporation during loading and electrophoresis. The SeqStudio performs the electrophoresis, separates the DNA fragments, and detects their fluorescence to generate a sequencing file. These files contain chromatograms—visual peaks representing each nucleotide—along with the base calls. The sequencing data is then transferred to a computer and analyzed using specialized software (such as Sequencing Analysis 7, or SeqA 7). The software allows users to visualize and interpret the sequence reads, identify mutations, and compare results with known sequences.

It is important to understand that Sanger sequencing is different from high-throughput or “next-generation” sequencing (NGS). While NGS can sequence millions of DNA fragments at once, it is more complex and expensive. Sanger sequencing remains the gold standard for smaller-scale applications, including verifying cloned genes, identifying point mutations, sequencing PCR products, or genotyping.

By the end of this lab, you will have a working understanding of how Sanger sequencing is performed from start to finish, including PCR setup, DNA purification, sequencing reaction, instrument operation, and data analysis. This hands-on experience provides a foundation for advanced studies in molecular biology, biotechnology, genetic engineering, and bioinformatics.

Reagents Functions and Purposes

Notes: DNA Sequencing (SeqStudio)

• CDC = Cathode Buffer Cartridge
  • Always use a new one for every sequencing run.
  • Remove the cartridge before turning off the instrument to prevent damage.
• You’ll prepare two samples per DNA:
  • One with forward primer. Do not combine Forward and Reverse.
  • One with reverse primer. Do not combine Forward and Reverse.
• SeqStudio uses 96-well plates
  • Each plate allows for ~25 injections (samples).
  • Fill all 4 lanes (machine runs 4 at a time), using Hi-Di Formamide in empty wells if needed.

Materials

• PCR reagents and thermal cycler
  • (Your target DNA, primers, dNTPs, polymerase, buffer)
• ExoSAP-IT
• Zymo ZR DNA Sequencing Cleanup Kit
• DNA-free water
• Nanodrop Spectrophotometer
• BigDye Terminator v3.1 Cycle Sequencing Kit
• Forward and Reverse primers
  • (Prepare separate tubes for each direction)
• PCR tubes (0.2 mL)
  • (For each sequencing reaction – 2 per sample)
• 96-well MicroAmp PCR plate
• Septa 8-strip caps
• Hi-Di Formamide
• Sequencing Standard (Optional Control)
  • (Mixed with BigDye Terminator v3.1)
• CDC Cartridge (Cathode Buffer Cartridge)
  • (Always use a new one for each run)
• SeqStudio Genetic Analyzer
• USB flash drive
  • (To export .ab1 files)
• Lab computer with Sequencing Analysis Software v7 (SeqA 7)

Procedure:

Day 1: PCR Product Cleanup with ExoSAP-IT

1. Run a PCR reaction for your target DNA using specific forward and reverse primers (This is the cDNA after RT-PCR)
   1. Obtain one PCR tubes with cap.
      • Label the tube clearly:
        • Primer gene names on the side
        • Initial or name on the top
   2. Add ___ µL of DNA (you need 200ng) into each PCR tube. 
      • Volume calculation for DNA Example: If you have DNA is 50 ng/µL → 200ng ÷ 50ng/µL = 4 µL needed
   3. Add enough water to bring the total to 12 µL (i.e., If 4 µL DNA, add 8 µL water)
   4. Into each PCR tube, use DIFFERENT pipet tips to add:
      • 10 µL of 2X Master Mix
      • 8 µL of your 2 µM primer Master (contains Reverse and Forward primers)
        • You should have 6 tubes in total, each having different primers
   5. Please place them in the thermal cycler (PCR machine), and set the machine to the following cycle:

Day 2: Part 1: Column-Based DNA Purification via Zymo ZR DNA sequencing Cleanup kit

1. After PCR, add 2 µL of ExoSAP-IT directly to each PCR tube.
   • Exonuclease removes leftover primers, and Shrimp Alkaline Phosphatase (SAP) inactivates excess nucleotides.
2. Incubate at 37°C (Waterbath) for 5 minutes to allow the enzymes to work.
3. Freeze at -80°C for at least 1 minute to stop the reaction and preserve the sample.
4. Add 240 µL of sequencing binding buffer to the PCR product.
5. Transfer this mix to the Zymo-Spin IB column over a collection tube.
6. Spin for 30 seconds to allow DNA to bind to the silica membrane.
7. Add 300 µL of sequencing wash buffer, spin again, and discard the flow-through in the sink.
8. Place the spin column in a clean 1.5 mL microcentrifuge tube.
9. Elute the DNA by adding 20 µL of DNA-free water and spin for 1 minute. Discard your spin column in the trash.
   • Now you have purified DNA in your centrifuge tube, ready for quantification.
   • Use a Nanodrop spectrophotometer to determine DNA concentration and record the value in nM/µL.
   • For sequencing, we aim for up to 100 ng of DNA per reaction.

Instrument Setup: Instructor-Only

Only trained personnel should prepare and initialize the SeqStudio machine.

1. Turn on the SeqStudio.
   • Login: Admin, Password: 1234.
2. Hit the eject button (top right) and lift open the door.
3. Retrieve the CDC cartridge from 4°C storage. Remove the plastic and protective capillaries and insert it into the instrument.
4. Close the door and wait for it to initialize.
5. Retrieve Hi-Di Formamide and Sequencing Standard BigDye Terminator v3.1 (BDT) from -20°C storage.
6. Mix 300 µL of Hi-Di Formamide into the sequencing standard BDT vial, if not already done.
   • Use a Sharpie and write "Hi-Di" on the BDT vial for future reference
7. During run setup: Choose "Sequencing → Plate", then select "ShortSeq (non-BDX)" with Dye Set: Z.Bigdye Terminator v3.1.
8. Hit eject (top right) to eject the plate
9. In one well, Instructor should add 20 µL of BDT + Hi-Di Formamide as a control.
10. All unused wells in the 96-well plate must be filled with 20 µL of Hi-Di Formamide OR the 20 µL of student's PCR sample to avoid run errors. The instrument injects 4 wells at a time, and it cannot process an empty one.
11. See the next step to prepare the student's PCR sample.

Day 2: Part 2: Sequencing Reaction Setup (Student's PCR Samples)

Each student will prepare two reactions per sample: one tube using the forward primer only and one tube with the reverse primer only.

1. Transfer up to 10 µL (or 100 ng) of your DNA to two new PCR tubes.
   • The maximum volume can be 10 µL, even if your DNA concentration is too low.
   • The minimum volume should be 2 µL, even if your DNA concentration is too high.
2. Add 4 µL of BigDye Terminator v3.1 to each tube.
   • The BigDye reagents incorporate labeled terminators that create a set of terminated fragments of various lengths, which the instrument can detect via fluorescence.
3. Add 4 µL of forward primer to one tube only, and 4 µL of reverse primer to the other tube only.
4. Adjust with nuclease-free water to bring the total volume to 20 µL.

Day 2: Part 3: Loading the SeqStudio

1. Place the samples in a MicroAmp 96-well tray retainer.
2. Cover with a septa 8-strip (single-use cap).
3. Load the plate into the SeqStudio.
4. Check the cartridge again to ensure it has enough buffer.
5. Retract the plate and start the run through the software.

Day 3:  Data Export and Analysis

Once the run is complete:

1. Export data to USB: Use the machine interface.
2. Plug USB into the lab laptop (Login: Username: Administrator, Password: Administrator).
3. Transfer data files from USB to the laptop.

Using Sequencing Analysis Software (SeqA 7):

1. Open SeqA 7. Login Password is SeqUser
2. Click File → Add Samples, navigate to USB, and add selected samples to import files.
3. After loading, click “Show Box” → go to the “Sequence” tab.
4. Copy your sequence into Notepad or another text editor for review.