SOLID sequencing | Supported oligonucleotide ligation and detection

Summary

This video explains the SOLiD sequencing method, which stands for Supported Oligonucleotide Ligation and Detection. The process begins with DNA amplification using emulsion PCR. The technique employs short 8-mer sequences with fluorescent probes to detect specific nucleotide pairings in DNA. The method records fluorescent signals and decodes nucleotide sequences by analyzing overlapping dinucleotide information. The video also covers how different fluorescent colors correspond to specific nucleotide combinations and demonstrates how overlapping signals help decode the entire DNA sequence. The detailed explanation concludes with a guide on how to interpret fluorescent signals during sequencing.

Takeaways

• 🔬 SOLiD stands for Supported Oligonucleotide Ligation and Detection, a sequencing method for DNA.
• 🧬 The DNA sample is first amplified using Emulsion PCR, and the beads of Emulsion PCR with the amplified product are used for sequencing.
• 🧩 SOLiD sequencing uses 8-mer probes that consist of two specific bases and six degenerate bases, allowing specific base pairing with the DNA template.
• 🌈 Different fluorescent probes are used based on the first two nucleotides in the sequence, with blue, green, yellow, and red representing different base pair combinations.
• 📸 A camera detects the color of the fluorescent signal after the probe binds, and this data is recorded for analysis.
• 🔄 After detecting the signal, the last three nucleotides are cleaved off, and the process repeats to read the next set of bases.
• 📜 The entire sequencing process involves multiple rounds using different primers, each with one fewer nucleotide than the previous primer (n-1, n-2, etc.).
• 🔍 The overlap of the last nucleotide from one round helps decode the next nucleotide sequence, ensuring accurate sequencing.
• 🧑‍🔬 By analyzing the recorded fluorescent signals, scientists can determine the correct nucleotide sequence of the sample.
• 📝 For exam preparation, start with explaining the full form of SOLiD, then describe the steps in the sequencing procedure, followed by the method of decoding the fluorescent signal.

Q & A

What does the term 'SOLiD' stand for in the context of DNA sequencing?

'SOLiD' stands for Supported Oligonucleotide Ligation and Detection, a method used in DNA sequencing.

What is the purpose of Emulsion PCR in the SOLiD sequencing process?

Emulsion PCR is used to amplify the DNA sample. The amplified DNA is attached to beads, which are then used for sequencing.

How does the 8-mer sequence function in SOLiD sequencing?

The 8-mer sequence contains two specific bases that pair with the template DNA and six degenerate bases that can pair with any nucleotide on the template.

What role does the fluorescent probe play in SOLiD sequencing?

The fluorescent probe indicates specific base pairings based on color. Each color represents a different dinucleotide combination, allowing for the identification of nucleotide sequences.

How are the colors of the fluorescent probes determined?

The colors are assigned based on the first two nucleotides of the sequence. For example, if both nucleotides are A, the probe is blue; if the first is A and the second is C, it is green; if the first is A and the second is G, it is yellow; and if the first is A and the second is T, it is red.

How does the SOLiD sequencing process detect specific sequences?

When the probe binds to the template, a DNA ligase enzyme ligates it to the primer. The fluorescent color of the probe is then detected by a digital camera, indicating specific nucleotide pairings.

What happens after the fluorescent signal is detected in the SOLiD sequencing cycle?

After detection, the last three nucleotides are cleaved off, removing the fluorescent tag. The cycle then repeats with new probes binding to subsequent sequences.

Why is the N-1 primer used in the SOLiD sequencing process?

The N-1 primer is used to analyze overlapping nucleotide sequences by removing one nucleotide from the original primer. This helps in decoding the sequence by revealing new overlapping pairs.

How is the fluorescent signal decoded to determine the DNA sequence?

Each fluorescent color represents four possible dinucleotide combinations. By using overlapping nucleotide information from successive probes, the correct sequence can be decoded.

Why is it important to have overlapping nucleotide information during decoding?

Overlapping nucleotides ensure continuity between detected sequences, allowing accurate determination of the DNA sequence based on the color-coded probes.