Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.
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ive been working in a lab for the past month using 3 different primers when i run pcr, but realized that i have no clue, aside that they have "complentary base pairs", on how exactly they break the genome into certain specific genes.
Can someone quickly explain it to me.
The primers are as u say a short designed complementary sequence, they come in pair, one forward (complementary to the non coding strand) and the reverse primer (complementary to the coding strand).
After the first heating step, the DNA separates and when the temp lowers down the primers anneal, forward and reverese on the two separate DNA strands. They make out the starting point for the polymerase (if they are not there the polymerase doesnt work, you can say it repairs the strand with the primer as the starting point). After two cycles the major produced DNA is the code between the primers.
So when u design primers u make them in a way that the gene u want is stuck between them. Thats why I asked why u used 3 primers.
Look in wikipedia.org for pictures, explaining it in just words are pretty hard.
Hope it cleared up alittle.
Your "forward" primer (generally) identifies the start or 5' region of DNA that is to be amplified.. wherease the "reverse" primer (generally) identifies where a region of DNA/gene ends or the 3' end of a DNA fragment.
In essence, the primers demark/identify the boundaries of DNA to-be-amplified (which can be a gene... or a gene/DNA fragment) during PCR.
So, if you use primers to amplify a gene... your primesr will amplify the sequence from beginning to end of the gene (5' to 3') which lies inbetween where the 2 primers anneal.
Thus, you can use primers to amplify virtually any gene or segment of DNA that you're interested in... as long as you have an idea of what the 5' and 3' boundary sequences are. (so that you can design appropriate primers).
Hope this helps ..
Maybe what's confusing you is how the polymerase "stops" replication of the desired fragment. Well..
_____________________________ if this is your double-stranded DNA from which you want to isolate a specific fragment, you'd first denature, so that the strands separate. Then, once you lower the temperature, the primers will anneal like this:
____________________________ and your polymerase will polymerize in the direction of the arrows.
The first time, your polymerase will polymerize until it falls off, but with further cycles of denaturing, annealing, and polymerization, you will end up with fragments the size of your desired genetic element plus the two primers on the ends.
6 posts • Page 1 of 1
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