For Sanger sequencing the pcr product can usually be sequenced with the pcr primers so generaly there is no difference between pcr and sequencing primers. For long products the pcr primers may only sequence part of the product so internal primers used only for sequencing can be used but in terms of size,sequence there is no real difference between the 2 types of primer. Sometimes you clone pcr product which has 2 advantages....first all sequencing can be done using the same plasmid sequence near each end of the inserted pcr product. Second your pcr product may have insertions,deletions or just 2 or more closely related sequences and cloning will allow sequencing of each unique sequence even when the sanger sequencing of the mixed pcr product can look lke a mess

In your earlier work you may just have needed sequencing of the same length amplimers and possibly were looking for base changes of other species in an environment which has mainly one species so changes in the sequence may just be SNPs which will show on an electropherogram as different from the usual sequence

You often want to transform your cloned product to not only store it, but to make sure that the plasmid can replicate in general. Transforming can also help you get more copies of the plasmid so when you prep it, you have more to work with for downstream applications like sequencing. Transformation can also be useful to isolate one cloned plasmid from another, something that may be necessary if off-target effects occur. You can then be confident that after sequencing, you know exactly what the sequence of your plasmid is.

1. Sequencing primers are short, single-stranded DNA sequences designed to bind specifically to the target region of DNA to initiate the sequencing reaction. They play a crucial role in DNA sequencing as they provide the starting point for DNA polymerase to begin synthesis. These primers are indeed used in DNA sequencing, particularly in methods such as Sanger sequencing and next-generation sequencing (NGS). Sequencing primers ensure the accurate reading of the DNA sequence by guiding the sequencing machinery to the correct location on the DNA template [1][2].

2. PCR products are sometimes cloned and transformed into competent bacterial cells to create a permanent, stable source of the amplified DNA. This step is necessary for several reasons:

- Stability and Quantity: Cloning allows for the production of large quantities of the DNA of interest. Directly sequencing PCR products might not provide enough material for repeated or extensive analysis.

- Error Reduction: PCR can introduce errors or produce mixed populations of DNA molecules, especially when dealing with complex samples. Cloning into bacteria allows for the isolation of single, uniform DNA molecules, reducing the risk of sequencing errors caused by PCR-induced artifacts [8][9].

- Ease of Manipulation: Cloned DNA can be easily manipulated, sequenced, and stored for long periods, facilitating further studies and applications [8].

Direct sequencing of PCR products is possible but may be less reliable due to the presence of mixed or erroneous sequences, especially in complex or high-throughput settings [3][5].

3. In your earlier works involving 16S rRNA PCR amplification, running a gel, excising the band, purifying it using a kit, and sending it for second-party sequencing is a common and effective workflow. This method helps ensure that the correct amplicon is sequenced by isolating the specific band corresponding to the 16S rRNA gene from the gel. This step is crucial for:

-Purity: Gel excision and purification remove non-specific products and primer dimers, ensuring that the sequenced DNA is the intended target [4].

- Accuracy: By isolating the correct band, you minimize the risk of sequencing errors and improve the accuracy of the microbial community analysis [4][7].

This workflow aligns with the need to obtain high-quality, specific sequences for accurate microbial profiling, as demonstrated in studies comparing different sequencing and analysis methods [1][2][6].

Reference

[1] Tremblay, J., Singh, K., Fern, A., Kirton, E., He, S., Woyke, T., Lee, J., Chen, F., Dangl, J., & Tringe, S. (2015). Primer and platform effects on 16S rRNA tag sequencing. Frontiers in Microbiology, 6.

[2] Schloss, P., Jenior, M. L., Koumpouras, C., Westcott, S. L., & Highlander, S. (2016). Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system. PeerJ, 4.

[3] Gloor, G., Hummelen, R., Macklaim, J. M., Dickson, R. J., Fernandes, A., MacPhee, R. A., & Reid, G. (2010). Microbiome Profiling by Illumina Sequencing of Combinatorial Sequence-Tagged PCR Products. PLoS ONE, 5.

[4] Nübel, U., Garcia-Pichel, F., & Muyzer, G. (1997). PCR primers to amplify 16S rRNA genes from cyanobacteria. Applied and Environmental Microbiology, 63, 3327 - 3332.

[5] O'Donnell, J. L., Kelly, R., Lowell, N., & Port, J. A. (2016). Indexed PCR Primers Induce Template-Specific Bias in Large-Scale DNA Sequencing Studies. PLoS ONE, 11.

[6] Xue, Z., Kable, M. E., & Marco, M. (2018). Impact of DNA Sequencing and Analysis Methods on 16S rRNA Gene Bacterial Community Analysis of Dairy Products. mSphere, 3.

[7] McGovern, E., Waters, S., Blackshields, G., & McCabe, M. (2018). Evaluating Established Methods for Rumen 16S rRNA Amplicon Sequencing With Mock Microbial Populations. Frontiers in Microbiology, 9.

[8] Finney, M., Nisson, P., & Rashtchian, A. (2001). Molecular cloning of PCR products.. Current protocols in molecular biology, Chapter 15, Unit 15.4 .

[9] Costa, G. L., & Weiner, M. (2006). Bidirectional cloning of PCR products.. CSH protocols, 2006 1.

Dear Dr. kishor kumar Keekan

1. Sequencing primers are used in the context of sequencing a DNA fragment with the intention of revealing its specific order of the nucleotide sequence. You can use PCR primers but just because these primers work in PCR reactions does not mean they’ll work for sequencing. There are certain limitations. For instance, a primer may start out mismatched against the template, but if even one primer manages to anneal, even briefly, the extension product will now have a perfect match and will amplify extremely well during the subsequent cycles. Sequencing reactions have no such benefit. With only one primer copying one strand, the reaction will always be less efficient for a mismatched primer.

Therefore, to obtain good sequencing results high quality primers and templates are important. Thus, when such primers are selected, they should be unique to a particular region where you desire to sequence. Sequencing primers are used in the context of sequencing a DNA fragment with the intention of revealing its specific identity. It also should be with a correct orientation where the sequences are usually generated from 3’ to 5’ ends of the primer. The sequence should be lacking undesirable self-hybridization such as the formation of hairpin loops. It should not contain consecutive formation of Guanine bases.

2. PCR products can be sequenced directly, and this will typically be much faster than cloning and sequencing. However, in certain cases, cloning first is either a requirement or at least beneficial. For instance, when your PCR cannot be optimized to generate a single product (even by gel extraction), direct sequencing will generate a messy data. Also, when templates contain difficult-to-sequence motifs (for example, homopolymers, microsatellites, high GC content), sequencing with cloned templates might help push through such trouble-spots.

Another case where cloning would be beneficial is when there are somatic mutations. The PCR products from genomic DNA preps will usually be a mix of slightly different sequences namely, various point mutations. Typically, each individual SNP will be represented at a very low level compared to the ‘consensus’ nucleotide at that position. In that scenario, if you directly sequence the PCR product, the resulting read will be the overall consensus and not the SNPs. Now when you clone the PCR product and sequence a single clone, the data will fix that fragment’s specific SNPs (if any exist) not the consensus sequence. So, it all depends on your experimental goal. If your goal is a consensus sequence, then direct sequencing would be better. However, if you are interested in ‘rare’ SNPs, then cloning would be required as rare SNPs would be drowned out by the consensus nucleotides from direct sequencing.

3. 16s rRNA sequencing is a method to identify and compare bacterial diversity from complex microbiomes or environments that are difficult to study. It is commonly used to identify bacteria down to the genus and/or species level. The protocols and methods used, however, vary considerably with regard to amplification primers, sequencing primers, sequencing technologies, as well as quality filtering and clustering. How results are affected by these choices, and whether data produced with different protocols can be meaningfully compared, is often unknown.

Regards,

Malcolm Nobre