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  #1  
Old 05-11-2011
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DNA PCR Primer Question

Hey fellas..have a doubt in PCR..its about the question in Kaplan biochem text..page 106..this the question they asked...

James k, is a Huntington disease patient who has recently been institutionalized and requires constant nursing care. His wife is unaffected, and they have a 38 year old daughter, Delia, who has 4 children ranging in age from 8 to 15 years. Delia wishes to know whether she has inherited the disease producing allele from her father. PCR amplification is carried out on the region containing the CAG repeat shown below. which of the following set of primers would be used to amplify the CAG repeat in the brackets (CAG) subscript n.

CTC AAG TCC TTC (CAG) SUBSCRIPT N CAA CAG CCG CCA


OPTIONS

A) GAG TTC AGG AAG n TGG CGG CTG TTG
B) GAA CTC CCT GAG n CCG CCA CCG CCG
C) GAA CTC CCT GAG n TGG CGG CTG TTG
D) CTC AAG TCC TTC n TGG CGG CTG TTC
E) CTC AAG TCC TTC n TGG CGG CTG TTG
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  #2  
Old 05-11-2011
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I am also confused about this, so thanks for bring it up.
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Old 05-11-2011
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Default here is what I think.

Is it E ?
primer has to be annealing the flanked region which is on 3' end and the sequence of which needs to be known as per requirement of PCR.
the primer is always complimentary to 3' prime borders sequence ( flanked region).
Let me know if its right or wrong, thanks.
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Old 05-11-2011
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yeh its E.

first thing, can primer be a DNA fragment. i have only heard of RNA primers being used for DNA synthesis.

2nd how is CTC AAG TCC TTC complimentary to

CTC AAG TCC TTC in

CTC AAG TCC TTC
(CAG) SUBSCRIPT N CAA CAG CCG CCA ??
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  #5  
Old 05-11-2011
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Quote:
Originally Posted by liaison4fun View Post
yeh its E.

first thing, can primer be a DNA fragment. i have only heard of RNA primers being used for DNA synthesis.

2nd how is CTC AAG TCC TTC complimentary to

CTC AAG TCC TTC in

CTC AAG TCC TTC (CAG) SUBSCRIPT N CAA CAG CCG CCA ??


you're looking at 5' end , which is the part to be amplified, you are not supposed to change it, just look at the 3' border, after the CAG subscript portion.
As for DNA primers, its mentioned in First Aid that they use DNA primers in PCR.
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Old 05-11-2011
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Quote:
Originally Posted by Sparco View Post
you're looking at 5' end , which is the part to be amplified, you are not supposed to change it, just look at the 3' border, after the CAG subscript portion.
As for DNA primers, its mentioned in First Aid that they use DNA primers in PCR.

ok so...u mean to say it sud be read like this

3' CTC AAG TCC TTC (CAG) SUBSCRIPT N CAA CAG CCG CCA 5'

for which the complimentary strand would be

5' GAG TTC AGG AAG (CAG) SUBCRIPT N GTT GTC GGC GGT 3'

SO SHOULDN'T 5' GAG TTC AGG AAG be the primer for

3' CTC AAG TCC TTC
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Old 05-11-2011
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I'll go with e
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  #8  
Old 05-11-2011
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We all know it is E from KLN note. The real queston is why? Can someone explain step by step pls?

why CTC AAG TCC TTC ?
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Old 05-11-2011
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Quote:
Originally Posted by liaison4fun View Post
ok so...u mean to say it sud be read like this

3' CTC AAG TCC TTC (CAG) SUBSCRIPT N CAA CAG CCG CCA 5'

for which the complimentary strand would be

5' GAG TTC AGG AAG (CAG) SUBCRIPT N GTT GTC GGC GGT 3'

SO SHOULDN'T 5' GAG TTC AGG AAG be the primer for

3' CTC AAG TCC TTC

NO, your question says

5' CTC AAG TCC TTC (CAG) SUBSCRIPT N CAA CAG CCG CCA 3'
NOW Look at the 3' side only in tha Question and make a complimentary strand (PRIMER) starting from the 3' end and write it in 5' to 3' direction which will be

5' CTC AAG TCC TTC (CAG) TGG CGG CTG TTG 3'
Stop before CAG , the Question is only asking about making of the primer which only requires the 3' border.
As I mentioned earlier, the segment before (CAG) has to be amplified , so we will not change it, we are only considering the flanked portion here that is the 3' border after the CAG subscript.
May be its my English that I'm not able to explain what I understand ,sorry about that, may be seniors can help elaborate the concept better

Last edited by Sparco; 05-11-2011 at 10:57 AM.
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  #10  
Old 05-11-2011
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Default

here's from the KAPLAN VIDEO lecturer:

CTC AAG TCC TTC (CAG) SUBSCRIPT N CAA CAG CCG CCA

we know that it is a single strand DNA and we must assume the left side is the 5' end and right side is the 3' end. now we're going to label it as strand 1 (5'-3' direction) and strand 2 (3'-5' direction).

<--------------------------------------------------PRIMER
strand 1: 5' CTC AAG TCC TTC (CAG)n CAA CAG CCG CCA 3'
strand 2: 3' GAG TTC AGG AAG (GTC)n GTT GTC GGC GGT 5'
PRIMER ------------------->

Now what we want to amplify is the portion in the parenthesis (CAG)n/(GTC)n,,we need primer. DNA SYNTHESIS is always 5'--> 3'


strand 2: 3' GAG TTC AGG AAG
primer: 5' CTC AAG TCC TTC =complimentary and anti-parallel
this amplify the (GTC)n

strand 1: CAA CAG CCG CCA 3'
primer: GTT GTC GGC GGT 5'
(read from R-L= TGG CGG CTG TTG)=complimentary and antiparallel
this amplify tremendous amount of (CAG)n

so answer would be:

E) 5' CTC AAG TCC TTC 3' n 5' TGG CGG CTG TTG 3'

Last edited by usmles99; 05-11-2011 at 11:19 AM.
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  #11  
Old 05-11-2011
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usmles99, Thank you very much for your clear and precise explanation! Really appreciated!
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