You can clone your PCR-product(s) and sequence individual clones.

Sanger sequencing is the best method to sequence. If your gene size is more than 600 bp then go for sanger sequencing with forward and reverse primers that will give you better results without any gaps. If size is much bigger then you may be require to design internal primers for sequencing apart from forward and reverse. Good luck.

Sabine Strehl's suggestion is the only way you can get useful information. You need to clone you PCR products, sequencing quite a few clones, and hopefully you will get all the possible alleles sequence information.

Yes clone your PCR products using plasmids.

Dear Mr Sabine and Zhang, Thank you and I agree with your answer, however, my sample size is at least 1000. cloning and sequencing these many samples (at least 5 per sample) will be too costly. That is why I want to know, whether there is any sequencing platform wherein if I give my heterozygous product that contains two types of DNA, will giove me two distinct reads of DNA?

A high quality Sanger sequence should be able to do this.  You can look at the traces quantitatively.  For example if you have an A/A, the A-trace should have a regularly shaped peak of a defined height and area.  If there's an A/G, then both the A and the G trace should peak at half the height / area.  You have to do some quality control to make sure your data quality is high enough to be able to make these calls reliably.  Feed it some known sequences, and see how reliably you can make these calls.  With highly pure samples and flawless execution, I think it's possible to get it to be reliable, especially if you know where to expect the differences.  

You can try pyrosequencing

If you have a sample size that large, the only reasonable thing to do is next generation amplicon sequencing (for example paired-end Illumina Myseq). For reviews on the methodology check:

Babik W, Taberlet P, Ejsmond MJ, Radwan J: New generation sequencers as a tool for genotyping of highly polymorphic multilocus MHC system. Molecular Ecology Resources 2009, 9(3):713-719.

Lighten J, van Oosterhout C, Bentzen P: Critical review of NGS analyses for de novo genotyping multigene families. Molecular Ecology 2014, 23(16):3957-3972.

Good luck! /Robert

If you do not want to or can not use sanger seq. for so many samples and pyroseq. is not available.....

In polyploid Phytophthora hybrids (Oomycete), an INRA-group used agarose-gel-based heteroduplex-analysis of cloned PCR fragmets for screening distinct alleles. Then they used sanger-seq for representatives of each allele-group identified by heteroduplexing.

Ioos, R. et al. (2006) Genetic characterization of the natural hybrid species Phytophthora alni as inferred from nuclear and mitochondrial DNA analyses. Fungal Genetics and Biology 43, 511–529

SSCP (single strand conformation polymorphism) may also be used for screening prior to sequencing.

Unfortunately, I do not know how costy and laborious these techniques are 

I agree with the answer of John Schloendorn, this option is, though tedious, the only low-cost way to analyze your samples. If you upload an example of your chromatograms, it would be easier to judge the quality for your sequence.

In the Sanger sequencing when you have a diploid base pair you will see that site has a doble pick in your chromatogram. You can confirm by seeing the same site in the other strand, this means that if you see the same doble pick in both strands is almost sure that you are in the presence of heterocigosis in that site. In that case you will need to code that site by using the IUPAC Nucleotide Ambiguity Code, where:

A: Adenine

C: Cytosine

G: Guanine

T (or U): Thymine (or Uracil)

R: A or G

Y: C or T

S: G or C

W: A or T

K: G or T

M: A or C

B: C or G or T

D: A or G or T

H: A or C or T

V: A or C or G

N: any base

. or - : gap

This is the less expensive way that you can proceed.

For a highly polymorphic, large and very complex multigene family like MHC I would really advice most strongly against using direct Sanger sequencing. As these genes are often duplicated you are likely to end up with more than two alleles per individual. Sanger sequencing such products directly will most likely give you an uninterpretable mess. If you really want to go for Sanger, then you must first separate the different alleles either using cloning or some gel based technique such as SSCP, DGGE or RSCP. However, with more than a thousand samples, such procedures will be very laborious. You are likely looking at years of lab-work doing optimisation and genotyping.

In contrast, using an amplicon next generation sequencing technique (MiSeq, 454 or similar), you will sequence each fragment independently (a bit like sequencing clones without doing cloning). With a sample size like yours, this is really the only reasonable way forward. It might also be worth starting by doing a small pilot on a handful of samples using traditional cloning just to get a feel for how variable and duplicated your loci of interest are.

The only cheaper option I can think about is to identify (from published sequences) genetic markers (microsatellites and SNPs) located within the sheep MHC, genotype these and use the genotypes a proxy for true MHC variation.

Dear all thank you very much.

I agree with the views of Robert Ekblom and similar other views.

Thanks Jorge,

however construction of algorithm requires a lot of information on available genotypes i guess.

isnt it?

Dear All

I am still unsatisfied with the way we handle MHC genotyping