cloning can solve many problems connected with fact that many genes in organism have multiple copies and pseudogenes (classic examle of multipe genes and pseudogenes is ribosomal cluster, which includes internal transcribed spacers - ITS1 and ITS2). In both cases in case of PCR without cloning you can observe all differences between all variatns becuse primers in most cases designed to coservative regions. So in some positions you can obtain doble or polysignal. Cloning makes possible to distingush different variats of gene and also pseudogenes and gives the possibility of further working with only 1 copy of gene.. So you can partially exclude infragenomic variation or to provide detailed study of it by repeating cloning many times. You can also find a lot of literature about why this technique is so impotrant. Some of them:

http://decapoda.nhm.org/pdfs/31071/31071.pdf

http://mbe.oxfordjournals.org/content/17/2/284.full.pdf

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0015854

cloning is necessary because when you sequence a nucleotide stretch if you use the primer which already used to amplify the gene you may not get first few and last few nucleotide sequence details accurately. so its better to clone the sequence in to a back bone vector (say pUC18) then amplify the sequence using primer sequences of the vector DNA. there you can be certain of full length sequence details are intact of the sequenced product. and also try to do bidirectional sequencing to minimize the error which may occur at either sides.

In my opinion you can directly sequence the PCR product if it was amplified from DNA of a single organism. If you have different products and want to do classical sequencing you have to clone so that you can sequence a single clone/PCR product.

cloning can solve many problems connected with fact that many genes in organism have multiple copies and pseudogenes (classic examle of multipe genes and pseudogenes is ribosomal cluster, which includes internal transcribed spacers - ITS1 and ITS2). In both cases in case of PCR without cloning you can observe all differences between all variatns becuse primers in most cases designed to coservative regions. So in some positions you can obtain doble or polysignal. Cloning makes possible to distingush different variats of gene and also pseudogenes and gives the possibility of further working with only 1 copy of gene.. So you can partially exclude infragenomic variation or to provide detailed study of it by repeating cloning many times. You can also find a lot of literature about why this technique is so impotrant. Some of them:

http://decapoda.nhm.org/pdfs/31071/31071.pdf

http://mbe.oxfordjournals.org/content/17/2/284.full.pdf

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0015854

Cloning is usually reserved for isolating a complex sequence (as mentioned), but is commonly also used to isolate a DNA fragment for which you do not have primers to amplify or sequence the DNA. A classic example of this is its use for sequencing of the human genome, where we did not know the sequence of the vast majority beforehand. Cloning enabled us to insert arbitrary DNA fragments of the human genome of approximately known size into vectors that had known primers sequences, primers which can then be used to direct DNA sequencing of the unknown fragment.

Refer to http://en.wikipedia.org/wiki/Molecular_cloning for more details.

In addition to all these answer, Cloning have other advantage you can preserved glycerol stock for future use.

You can sequencing before cloning as well as after cloning. It is not necessary to get sequence after cloning, it will be advantageous. If you want to study of a gene or gene products, you sequence them before cloning but some times you doesn't get the protein expression. During the cloning some times you get mutations or incomplete sequence cloning, so after cloning sequence make the thing clear.

Hi Kinjal,

You can either clone and sequence or you can sequence directly. I donot think cloning is absolutely necessary for the sequencing. But before that I would recommend you to check the amplified product in the gel and if there are multiple bands, gel elute your product of interest.

Best regards.

I assume that you're amplyfing from one individual or from something that is genetically homogeneous. I think that a good solution is to sequence directly the pcr product from 3 indipendent amplification reactions using both forward and reverse primer (6 sequences). If you find that your sequence is not from a unique amplicon (i.e. you see double peaks or you have partially or completely unreadable electropherograms) then you need to clone. Otherwise, if your sequence is ok then you can assemble fw sequence and reverse sequence (assuming that your pcr product is not bigger than 1000-1200 base pairs, otherwise you could have a gap in the middle). Then you can align the sequences of your 3 indipendent pcr reactions and when you find a difference in a particular position for only one out of three of them discard it (i.e. T/T/A, keep T and discard A). Such differences are likely due to pcr artifacts.

I believe one issue may be missing from this conversation. While I agree with Andrii, that cloning will assure a single sequence (provide a single sequence for multi-copy genes), this could be a bad thing. You are trowing away information. I do not know much about zoanthids, but I assume they have mitochondria that contain many many genome copies, and it is quite possible that they are heteroplasmic. If you clone, you lose all information regarding the possible heteroplasmy. Further, it is possible that by chance (or simple bad luck), that you clone and sequence a specific gene copy that may be fairly rare. I would strongly advise a protocol such as Francesco describes where you sequence PCR products from a number of independent reactions. I disagree somewhat with Francesco about what to do if you find non resolved bases in your sequencing. It is quite possible that these are not PCR errors, but rather result from the presence of multiple, distinct genome copies in the mitochondrium. Especially in the case of bar-coding, it vital that you know if there is any sequence variation among members of a species, or even within a single individual.

Cloning also ensure that you have a cloned copy of the gene available to use in PCR. We actually sequence our PCR products directly if it targeted gene sequences i.e. ITS, RPB2. But I will still clone one of the amplicons for future use in PCR tests.

After cloning sequencing is necessary because to confirm that whatever sequence we clone (which gives expression) if that sequence is present in sequencing data then we can say that our cloning results are true .