Different organisms have specific sequences that are particular to only one type of oraganism or that help us differentiate among organisms. Some example of such repeats are VNTRs(Variable number tandem repeats), SNPs( single nucleotide profiling).These sequence help us create a genome profile for organisms and helps us to group organisms or humans based on the type of genome profile. Among many others, AFLP(Amplified fragment length polymorphism) is one such technique that helps us identify such repeats.
WHAT IS AFLP?
AFLP or Amplified Fragment Length Polymorphism is a PCR based tool used in molecular diagnostic techniques. It was developed in the early 1990s by Keygene. AFLP are DNA fragment obtained after digestion by restriction enzymes . The ligation products are then digested to oligonucleotide adapters followed by selective amplification using PCR. This is the reason, AFLP technique is known to be a combination of RFLP and PCR.
Selective Fragment Length Amplification (SFLA) and Selective Restriction Fragment Amplification (SRFA) are synonyms sometimes used to refer to AFLPs. The PCR primers consist of core sequences that are part of the adapters, restriction enzyme specific sequence and 1-5 selective nucleotides. The higher the number of selective nucleotides lower will be the number of bands obtained.
The AFLP technology has the capability to detect various polymorphisms in different genomic regions simultaneously. It is also highly sensitive and reproducible. As a result, AFLP has become widely used for the identification of genetic variation in strains or closely related species of plants, fungi, animals, and bacteria. The AFLP technology has been used in criminal and paternity tests, also to determine slight differences within populations, and in linkage studies to generate maps for quantitative trait locus (QTL) analysis.
The technique was originally described by Vos and Zabeau in 1993.In detail, the procedure of this technique is divided into three steps:
- Digestion of total cellular DNA with one or more restriction enzymes and ligation of restriction half-site specific adaptors to all restriction fragments.
- Selective amplification of some of these fragments with two PCR primers that have corresponding adaptor and restriction site specific sequences.
- Electrophoretic separation of amplicons on a gel matrix, followed by visualization of the band pattern.
- RESTRICTION DIGESTION
At first the sample of DNA are cut using restriction enzymes. Depending on the number of restriction sites in the DNA samples restriction fragment are produced . For example, if a sample 1 has DNA with one restriction site for EcoR1 then it will produce only 2 fragment, and if sample 2 has DNA with restriction sites for EcoR1 then it produces 3 fragment.
2. ADAPTOR ADDITION AND SELECTIVE AMPLIFICATION
Once the restriction fragments are generated the next step involves amplification of these fragments. But if we directly go for PCR the reaction may face a problem as the restriction digestion creates sticky ended restriction fragments. So, during PCR there is probability that the primers may bind to the sticky ends. To avoid this, to the DNA fragments adaptors are added which convert the sticky end to blunt ended fragments.
In the next steps PCR amplification is performed and primers with core sequences that are part of the adaptors are added.
Preamplification- This first amplification that uses the adaptor sequences as primers allows only those fragments to be amplified that have adaptors ligated to their extremeties. Additionally to adaptor sequences, the primers used for pre-selective amplification also have an supplementary base. This extra base allows another selection that amplifies 1/4 th of the fragment that have adaptors ligated at the extremeties. Till here the DNA can be visualized on agarose gel.
Selective amplification- The aim of this step is to restrict the level of polymorphism and to label the DNA. For this second amplification 3 more nucleotides are added at 3′ end of the primer sequence that is used in preamplification. These additional nucelotides make amplification more selective and decrease the number of restriction fragments amplified. Also, in this step one of the primers is labelled with florescent dye that helps in visualizing the DNA during migration. The PCR products are visualized using acrylamide gel electrophoresis.
3.POLYACRYLAMIDE GEL ELECTROPHORESIS
Once the PCR is completed the amplified fragment are run on polyacrylamide gel and DNA are separated based on their charge and mass. The bands are then visualized and different band samples compared and studied for polymorphism.
25 thoughts on “AMPLIFIED FRAGMENT LENGTH POLYMORPHISM”
The information is sufficient to clear my doubts about pre selective amplification and selective amplification. Thank you Mrs Aswathi
Good post however I was wondering if you could write a litte
more on this subject? I’d be very grateful if you could elaborate a little bit further.
Long time supporter, and thought I’d drop a comment.
Your wordpress site is very sleek – hope you don’t mind me asking what theme you’re using?
(and don’t mind if I steal it? :P)
I just launched my site –also built in wordpress
like yours– but the theme slows (!) the site down quite a bit.
In case you have a minute, you can find it by searching for “royal cbd”
on Google (would appreciate any feedback) – it’s still in the works.
Keep up the good work– and hope you all take care of yourself during the coronavirus
Nice blog here! Also your site loads up fast! What host are you using?
Can I get your affiliate link to your host? I wish my website loaded up as fast as yours lol
Very rapidly this web site ᴡill be fqmous amid аll blog usеrs, due to іt’s fastidious ϲontent
Hello! I fair-minded would like to afford a mammoth thumbs up recompense the gargantuan info you contain here on this post. I solution be coming resile from to your blog also in behalf of more soon.
Good answers in return of this matter with firm arguments and explaining all concerning that.
It’s a shame you don’t have a donate button! I’d definitely donate to this brilliant blog!
I guess for now i’ll settle for bookmarking and adding
your RSS feed to my Google account. I look forward
to brand new updates and will talk about this blog with my Facebook group.
Hi, I do believе thіѕ is an excellent website. I stumbledupon іt ;
) Ι may cօme ƅack onc again sіnce I bookmarked it.
Monhey and freedom іѕ the best wаy to cһange, may you be rich and cntinue too
guide otheｒ people.
Greetings from Florida! I’m bored to tears at work so I decided to browse
your site on my iphone during lunch break. I really like the
information you provide here and can’t wait to take a look when I get home.
I’m surprised at how quick your blog loaded on my mobile ..
I’m not even using WIFI, just 3G .. Anyways, fantastic site!
great put up, very informative. I’m wondering
why the opposite specialists of this sector don’t notice
this. You should proceed your writing. I am sure, you’ve a great readers’ base already!
This piece of writing offers clear idea in favor of the new viewers of blogging,
that truly how to do running a blog.
you’re іn point of fact a good webmaster. The website loading speed iis incredible.
Ӏt seems that уοu’re doing any unique trick. In addition, The
сontents are masterwork. you’ve performerd a
exzcellent job οn tһiѕ topic!
I do believe all the ideas you have introduced on your post.
They are very convincing and will certainly work.
Nonetheless, the posts are too brief for starters.
May just you please lengthen them a little from subsequent time?
Thanks for the post.
For mоst recent inf᧐rmation you hаve to pay a visit
web and on internet I found tһіѕ site aѕ а finest web
site for hottest updates.
Tһis article іѕ trulʏ a nice one іt helps new tһe web visitors, ԝho arｅ wishing in favor of blogging.
Like!! I blog frequently and I really thank you for your content. The article has truly peaked my interest.
Really enjoyed this article post.Really looking forward to read more. Fantastic.
Hey! This is my first comment here so I just wanted to give a quick shout
out and say I really enjoy reading through your blog posts.
Hello there, You have done an excellent job.
I will certainly digg it and personally suggest to my friends.
I am sure they’ll be benefited from this web site.
adreamoftrains web hosting providers
Hello, i think that i saw you visited my site thus i came to return the
prefer?.I’m trying to in finding things to improve my website!I guess its adequate to use a few of your concepts!!
Excellent website. Plenty of helpful info here.
I am sending it to some buddies ans additionally sharing in delicious.
i like this excellent article