phuluong2k
Forum Fanatic
Topics: 714
Posts: 2,008
|
Using a primer that binds to the 3' flanking region on the coding strand to initiate the replication of HFE gene and radiolabeled dideoxynucleotides to terminate synthesis, he obtain the result shown on the sequencing gel below
http://www.geocities.com/phuluong2k/Q1.gif
Which of the following is the sequence of primer extension ?
A. ACG ACG TTA CTG GC
B. CTG TCA TTG CAG CA
C. GAC AGT AAC GTC GT
D. TGC TGC AAT GAC AG
plz give explanation
|
| Frida
Forum Newbie
Topics: 0
Posts: 4
|
This question was also in the Q-bank. But with a twist, it asked tor the HFE gene coding strand:
CTG TCA TTG CAG CA.
The sequence gel is read from bottom to top (short to long) this will give you directly the sequence of the primer extension:
TGC TGC AAT GAC AG
When you want to know the coding strand (more likely in real life) not the primer sequence you have to ID the primer extension sequence, the coding strand will be the sequence complementary-antiparallel to this one. This is a primer that start in the 3'end of the coding strand. The coding strand would be complementary and antiparalle to the primer extension.
Usually a primer sequence is known. You buy it or synthesize it in the lab. It is usually "hot". The sequence you are interested in is the complementary and antiparallel to this primer. Most important, you are interested in the hundred-thousand nucelotides that follow the primer sequence after amplification. This will give you the nucleotides you don't know in the coding strand.
When you have question like this, shorter your options by
1. identifying the first nucleotide (bottom)
2. Which strand you are looking for
if is primer sequence read as it is -complementary AND parallel-
if it is a coding strand DNA -complementary AND antiparallel to primer sequence-
|
| phuluong2k
Forum Fanatic
Topics: 714
Posts: 2,008
|
Thank for explanation, exactly it is in the Qbank
i can study by heart the the primer sequence is from the bottom to the top ( short to long ) but can you explain why we have this result. Plz clarify a little more, thanks
|
| phuluong2k
Forum Fanatic
Topics: 714
Posts: 2,008
|
What does it mean short to long ?
|
| Frida
Forum Newbie
Topics: 0
Posts: 4
|
What you are looking @ in the question is a "chain termination" method of sequence DNA.
Bottom =short chain to Top =Long chain
In the termination method, DNA is replicated by using polymerase enzymes to extend a primer attached to the sequence of interest. Dideoxynucleotides added to the mixture are incorporated into the growing strand but terminate the replication process as they can have no more bases added to them. On completion of the reaction a population of products is generated. All products start with the primer and terminate with the dideoxynucleotide, the difference being in the position at which the terminator is included. This results in the strands being different lengths.
The reaction is repeated four times; each time a different base being present as the dideoxynucleotide. The products are then separated on different lanes by gel electrophoresis to read the sequence of bases. The gels then had to be analysed by hand, a long and repetitive job.
Nowdays there are fluorescent probes and computer programs that make the dream of sequence a true real posibility.
|
| phuluong2k
Forum Fanatic
Topics: 714
Posts: 2,008
|
Thanks alot, I am clear now
|
|
| |
| | | | | | |
