Sticky ends and blunt ends in DNA cloning?

[sameeralord]

1. Ok in the plasmid we used the ECOR1 to create sticky ends. Now my question is when we add DNA ligase, why don't these ends stick back again. How are we able to add new DNA piece in between. Basically what I'm saying is when ECOR 1 creates sticky ends in plamids, why do they separate the plamid, don't the sticky ends stick back together.

2. Also why can't DNA ligase bind the blunt ends, can't it fix the phosphodiester bond there.

Thanks

 

[Ygggdrasil]

1. Ok in the plasmid we used the ECOR1 to create sticky ends. Now my question is when we add DNA ligase, why don't these ends stick back again. How are we able to add new DNA piece in between. Basically what I'm saying is when ECOR 1 creates sticky ends in plamids, why do they separate the plamid, don't the sticky ends stick back together.

It is possible for the sticky ends of the plasmid to come back together. In fact, re-ligation to recover the original plasmid is more likely in the case shown above than insertion of the purple DNA into the plasmid.

Molecular biologists overcome this problem in one of two ways. The first way is to treat the plasmid with an enzyme called a phosphatase. The phosphatase removes the 5' phosphate groups from the plasmid DNA. Because the 5' phoshphates are required for DNA ligase to create a new 5'-3' bond, phosphatase treatment prevents the plasmid from re-ligating. Because the purple DNA was not treated with phosphatase, it still retains 5' phosphate groups, so ligase can still join the 5' ends of the purple DNA to the 3' ends of the plasmid. This procedure results in a plasmid with two nicks (because the 5' ends of the plasmid cannot join to the 3' ends of the purple DNA). However, when you put the DNA back into a bacterium, enzymes inside of the bacteria are able to join these ends to create a fully ligated plasmid.

The second solution is to use two different restriction enzymes to cut the plasmid and purple DNA. This gives two incompatible sticky ends on the plasmid that cannot bind together and be ligated. Furthermore, this allows one to ensure that the purple DNA inserts into the DNA in a defined orientation (whereas if you use only one restriction enzyme as in the example you gave, the purple DNA could insert in one of two different orientations).

2. Also why can't DNA ligase bind the blunt ends, can't it fix the phosphodiester bond there.

DNA ligase can join blunt ends. This process, however, is much less efficient because the blunt ends are not bound together as sticky ends are. Furthermore, molecular biologists like to use sticky ends because these provide specificity to the ligation. Ligase will join DNA fragments with compatible sticky ends and will not join fragments with incompatible sticky ends. In blunt end ligations, you have little control over which blunt ends ligase joins together.

 

[sameeralord]

Thanks Ygggdrasil. That was just what I was after. Thanks for the great answers
If you could can you send me a link to a pic where they use 2 restriction enzymes, to insert dna. Thanks

 

[Ygggdrasil]

Here's an image:
http://web.virginia.edu/Heidi/chapter13/Images/8883n13_06.jpg

 

[sameeralord]

Here's an image:
http://web.virginia.edu/Heidi/chapter13/Images/8883n13_06.jpg

Thanks Ygggdrasil Just a quick question. In your previous post you said that when one restriction enzyme is used, the dna can be joined in 2 different ways. I see, when you turn it upside down you can do it, but what is the disadvantage in this, isn't it still the same.

 

[Ygggdrasil]

Let's say you want to express a protein from the plasmid you're creating. If the insert DNA goes in the wrong way, you the plasmid won't code for the protein you want.

 

[sameeralord]

Let's say you want to express a protein from the plasmid you're creating. If the insert DNA goes in the wrong way, you the plasmid won't code for the protein you want.

But since the DNA region is a palindrome, wouldn't it be exactly the same the other way round.

 

[Ygggdrasil]

The short ~6 nucleotide cut site recognized by the restriction enzyme is palindromic, but the sequence between the two cut sites is not necessarily palindromic. Consider this simple example. The DNA you want to insert has the sequence:

5' - GAATTCAAAAAAAAAAAGAATTC - 3'
3' - CTTAAGTTTTTTTTTTTCTTAAG - 5'

Where the underlined sites represent the EcoRI sites. Now let's say the plasmid will express protein from the top strand of the plasmid. Depending on what orientation the insert DNA goes in, you will either insert a stretch of As or a stretch of Ts, which would code for different amino acids.

 

[sameeralord]

The short ~6 nucleotide cut site recognized by the restriction enzyme is palindromic, but the sequence between the two cut sites is not necessarily palindromic. Consider this simple example. The DNA you want to insert has the sequence:

5' - GAATTCAAAAAAAAAAAGAATTC - 3'
3' - CTTAAGTTTTTTTTTTTCTTAAG - 5'

Where the underlined sites represent the EcoRI sites. Now let's say the plasmid will express protein from the top strand of the plasmid. Depending on what orientation the insert DNA goes in, you will either insert a stretch of As or a stretch of Ts, which would code for different amino acids.

Oh I get it now. Thanks! Another quick question. What does it mean when they human genome is exremely heterogenous and cDNA is hetergenous. I know what heterogenous means in terms of alleles like aa, but I don't think they are talking about that in this case.

 

[Ygggdrasil]

I'm not sure. Do you have the context for this statement?

 

[sameeralord]

I'm not sure. Do you have the context for this statement?

2. Complementary DNA (eDNA) libraries: If a gene of interest is
expressed at a very high level in a particular tissue, it is likely that
the mRNA corresponding to that gene is also present at high con-
centrations in the cell. For example, reticulocyte mRNA is com-
posed largely of molecules encoding the (-globin and I-globin
chains of hemoglobin. This mRNA can be used as a template to
make a complementary double-stranded DNA (cDNA) molecule
using the enzyme reverse transcriptase (Figure 32.7). The result-
ing cDNA is thus a double-stranded copy of mRNA. cDNA can be
amplified by cloning or by the polymerase chain reaction. It can
be used as a probe to locate the gene that coded for the original
mRNA (or fragments of the gene) in mixtures containing many
unrelated DNA fragments. If the mRNA used as a template is a
mixture of many different species, the resulting cDNAs are hetero-
geneous. These mixtures can be cloned to form a cDNA library.
Because cDNA has no intervening sequences, it can be cloned
into an expression vector for the synthesis of eukaryotic proteins
by bacteria (Figure 32.8). These special plasmids contain a bac-
terial promoter for transcription of the cDNA, and a Shine-
Dalgarno sequence (see p. 435) that allows the bacterial
ribosome to initiate translation of the resulting mRNA molecule.

 

[Ygggdrasil]

In this context, it just means that the cDNAs are different from each other (correspond to different mRNA sequences).

 

[jashanjot]

cud u please explain me as to how we decide that enzymes that produce sticky ends or blunt ends must be put in the dna to be inserted... how is it decided... reply as soon as spossible cos i need to design the primers for inserting my fragment in the pritein expression vector... i want to see expression of intersted protein and also clone it.. in the same vector..
also i need to know that i want to take out my protein form the palsmid and want to do further other experiments.. so how shall i plan to select the enzymes for the same

hope to hear form sameeralord or Ygggdrasil... cos i liked ur discussion and i want to understand more about the same cos i am new to molecular stuff,, thank you

 

[jashanjot]

cud u please explain me as to how we decide that enzymes that produce sticky ends or blunt ends must be put in the dna to be inserted... how is it decided... reply as soon as spossible cos i need to design the primers for inserting my fragment in the pritein expression vector... i want to see expression of intersted protein and also clone it.. in the same vector..
also i need to know that i want to take out my protein form the palsmid and want to do further other experiments.. so how shall i plan to select the enzymes for the same

hope to hear form sameeralord or Ygggdrasil... cos i liked ur discussion and i want to understand more about the same cos i am new to molecular stuff,, thank you

please reply me as soon as possible

 

[Monique]

Sticky is always better than blunt, just have a look at the enzymes that are in your vector and design a primer based on that. Pick an enzyme that is in the middle of the multiple cloning site, so that you do not remove all the enzyme recognition sites with your first experiment.