I am currently working on DRD4 exon 3 VNTR to analyse association with methamphetamine abuse.Actually I have two questions-
1) I am following several papers.According to the paper the fragment size after gel electrophoresis ranges from 500 to 750 basepairs(I attached the gel picture from the paper)but after gel electrophoresis I got totally different banding pattern( I also attached my gel picture).
What is the reason behind this difference?I used the same primers mentioned in the paper.
In the paper they also mentioned denaturing polyacrylamide gel electrophoresis.I am a little bit confused about it.
2) secondly I can not understand the analysis result as 2/2 repeat type,2/4 repeat type,$/4 repeat type,4/5 repeat type,4/7 repeat type-these are mentioned as the analysis result.Have anyone worked on this previously? I also attached another picture regarding this analysis result of different repeat type in capture 3.
it looks like there is about a 25bp repeated motif and the naming is just the number of repeats so 2,4 is 2x25 and 4x25 bases of repeat in the amplimer on each chromosome
The reason that your distribution is different is probably just that some alleles are more common in different populations and your population produces different distributions of each allele size because mainly the distribution is random unless selection or inbreeding concentrates some alleles. Denaturing PAGE is just the addition of urea or some denaturant to simplify the structure of the dna amplimer so that all molecules are linear so will run in the gel according to their size and not according to their shape ( as in non denaturing PAGE)
Paul Rutland Thank you so much for your information.I decided to try denaturing PAGE next time to avoid any secondary structure formation of DNA.
Would you please tell me -how they are detecting that band at 500bp (basepair) indicates 2 repeats (550bp,600bp,700bp indicates 3,4,6 repeats respectively) ? (if possible) Should I have some knowledge on sequencing to understand this? is there any other easy way to understand how the repeat number is assesed from fragment size?
I am afraid how will I interpret a band between 100 and 200 bp as I cound not find any interpretation for this banding pattern.Again gene sequencing is currently not possible in the laboratory I am working.
Thank you.
Hi Sonia,
When the authors designed their pcr primers they had the gene sequence available so they will have designed primers that would have amplified a 400 base fragment if there were no repeat sequences between the primers. Then when they amplify the fragment any size increase must be caused by repeat sequences between the primers . One repeat is 50bp so an increase in size of 100 means 2 repeats etc. No sequencing is needed just an accurate assessment of band size.
The small band at 150 bases is probably caused by primers annealing in the wrong place due to too low an annealing temperature in the pcr reaction and giving a band from some other part of the genome. It can be ignored and is not relevant to the repeat fragments but it does indicate that your pcr is not well set up so I would run a temperature gradient pcr to get a cleaner product which will also increase the intensity of your specific bands, If you do not have a gradient pcr machine then just try increasing the annealing temperature in 2 degree steps and see if your specific bands get stronger and the 150 band gets weaker
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