Question

I've been looking for methods for immobilizing 5'-Thiol modified dsDNA on gold slides. Most of what I've come across suggests using DTT followed by a desalting step or using TCEP. However, I haven't had any luck reducing and then conjugating the DNA onto the gold slides. I've seen some methods use DTT, and some use TCEP, and some include an incubation step with MCH. It's been difficult finding a complete and satisfactorily detailed method online.

The DNA I am using is a 1 kb long double-stranded molecule with a 5'-Thio modification at one end and a 5'6-FAM modification at the other end. I need to synthesize the strands myself via PCR using two modified primers and a template strand rather than ordering the fully modified 1 kb strand, so my working concentrations are usually fairly low (~70-100 ng/uL after PCR and spin-column purification).

Can anyone provide a detailed method that has worked for them for reducing thiol modified DNA and immobilizing it onto gold surfaces or gold monolayers?

Answer 1

To immobilize 5'-Thiol modified dsDNA on gold slides, you can use methods such as reducing the thiol groups with DTT or TCEP, followed by desalting. Another method is to use MCH during the conjugation step. Working concentrations of the thiol modified DNA should be low, and each step needs to be optimized for successful immobilization.

Step-by-step explanation:

Immobilizing 5'-Thiol modified dsDNA on gold slides can be achieved using different methods. One approach is to use DTT (DL-dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine) to reduce the thiol groups, followed by a desalting step to remove any unreacted reducing agent.

Another method involves using MCH (2-mercaptoethylamine hydrochloride) during the conjugation step. The working concentrations of the thiol modified DNA are usually low, so it is important to optimize the conditions for immobilization.

Here is a detailed method that you can try:

1. Prepare 5'-Thiol modified dsDNA by synthesizing the strands using PCR with modified primers and a template strand. Purify the DNA using a spin-column purification method.
2. Reduce the thiol groups on the DNA by incubating it with DTT or TCEP. Use a suitable concentration of the reducing agent and optimize the incubation time.
3. Remove any unreacted reducing agent and salts by desalting the DNA. This can be done using a desalting column or by dialysis.
4. Prepare the gold slides or gold monolayers by cleaning them thoroughly to ensure optimal surface binding.
5. Conjugate the reduced thiol modified DNA to the gold surfaces. This can be done by incubating the DNA with the gold surfaces in the presence of MCH or other suitable conjugation reagents. Optimize the conditions for conjugation based on the DNA concentration and incubation time.
6. Wash the gold surfaces to remove any unbound DNA and other impurities.
7. Verify the immobilization of the DNA on the gold surfaces using appropriate characterization techniques such as fluorescence microscopy or surface plasmon resonance.

Remember to keep the working concentrations of the DNA low and optimize the conditions for each step to ensure successful immobilization onto gold surfaces.