DNA - Sticky and blunt ends- Explain

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pyrophoenix
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DNA - Sticky and blunt ends- Explain

Post by pyrophoenix » Thu Oct 19, 2006 8:14 pm

I am a first yr Biotechnology student and I did not understand the meaning of the terms Blunt and sticky ends! I do know that when an endonuclease clevages the DNA opposingly it is blunt and if not opposite it is sticky ..But the problem is that I dont understand what it means!
More over what are all the letters

"A A G C TT " stand for?
How are they helpful.. What is their purpose?
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pyrophoenix
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reply to my query !

Post by pyrophoenix » Thu Oct 19, 2006 8:18 pm

Please if you are kind enough... Please send me an Email regarding the answer!
Thanks a lot!
My id is

[email protected]
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G-Do
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Post by G-Do » Thu Oct 19, 2006 8:46 pm

A DNA molecule is a sequence of smaller molecules called nucleotides that have been daisy-chained together, end-to-end. There are four kinds of nucleotides in a DNA molecule: adenine, guanine, cytosine, and thymine. These are abbreviated to A, G, C, and T. So to do an example, a single molecule of DNA could look like:

ACCGATTAGCGAC

- which is an adenine, attached to a cytosine, attached to another cytosine, attached to a guanine, and so on.

In cells, DNA molecules tend to occur as double-stranded dimers. That means that there will be two chains, or strands:

ACCGATTAGCGAC and TGGCTAATCGCTG

- and these two strands will attach to each other longways:

ACCGATTAGCGAC
TGGCTAATCGCTG

Each of the nucleotides in the upper strand attacheds specifically to its partner in the lower strand. This phenomenon is known as "base-pairing" and it is achieved chemically through the formation of hydrogen bonds. However, not all DNA strands may attach - they must obey certain rules. The rules for base-pairing are: A may only attach to T, and G may only attach to C. If you examine the two strands above, you'll see that whenever an A is on top, a T is on bottom (and vice versa) and whenever a G is on top, a C is on bottom (and vice versa).

Now, an endonuclease is an enzyme (a protein or an RNA) which clamps onto the double strand like a claw and slides around until it finds a specific sequence of As, Ts, Gs, and Cs. For example, an endonuclease might recognize ATTA on one strand. When it finds the sequence it needs, it cuts both strands at that spot. In other words, if our example endonuclease (represented by an *) were to be mixed with our toy DNA sequence, it would slide along the strand like this until it hits the ATTA in the top strand:

*
ACCGATTAGCGAC
TGGCTAATCGCTG

--*
ACCGATTAGCGAC
TGGCTAATCGCTG

-----*
ACCGATTAGCGAC
TGGCTAATCGCTG

---------*
ACCGATTAGCGAC
TGGCTAATCGCTG

------------[*]
ACCGATTAGCGAC
TGGCTAATCGCTG

Endonucleases can cut in one of two ways. Either it makes a clean cut or a jagged cut. A clean cut is something like this:

------------[*]
ACCGAT-----TAGCGAC
TGGCTA-----ATCGCTG

You can see that there is no overhang on the top or the bottom strand. When the endonuclease makes a clean cut, the two ends of the new molecules are called "blunt ends." On the other hand, a jagged cut is something like this:

------------[*]
ACCGATTA---------------GCGAC
TGGC---------------TAATCGCTG

You can see that there is an overhang of four nucleotides on the top of the left-hand DNA molecule and also on the bottom of the right-hand DNA molecule. These ends are known as "sticky ends" because they are free to attach themselves to whatever oligonucleotides are floating around in solution. Sticky ends are useful in that they allow you to clone DNA into a plasmid very easily - just make complementary stick ends on your plasmid, and the DNA will fit right in.

I hope this has made things clearer.
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pyrophoenix
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Thanks a lot!

Post by pyrophoenix » Fri Oct 20, 2006 12:13 pm

Hey thank you very much! you were a great help! Thanks!
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pyrophoenix
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more questions!

Post by pyrophoenix » Fri Oct 20, 2006 2:47 pm

How or what are the factors that determine whether a DNA cleavages with Jagged ends or Smooth ends?
please let me know!
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mith
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Post by mith » Fri Oct 20, 2006 4:10 pm

I think it has to do with which endonuclease you use
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MrMistery
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Post by MrMistery » Fri Oct 20, 2006 6:14 pm

yeap, yeach endonuclease has a specific "clipping pattern". the enzyme will always cut the DNA in the same way, regardless of the environment around it etc. that is why we get complemetary sequences when we cut the gene of interest and the vector with the same restriction endonuclease.
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