NOVA Online | Cracking the Code of Life

Sequence for Yourself
Part II: Cloning
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Mix

Mix Human Fragments to Vectors
To clone DNA, we isolate the fragments from one another, then make many copies, or clones, of each one of the fragments. This work is most often done in bacterial host cells.

We have mixed our DNA fragments with cloning vectors. (A vector is a short piece of DNA that is capable of replicating on its own when inside a cell.) Vectors are usually circular, but these have been cut open by a restriction enzyme.

Fragments and Vectors Combine
With the help of a DNA ligase, which is an enzyme that helps to connect pieces of DNA, the human DNA fragments and the DNA of the vectors combine.

The vectors have become circular again.

Vectors

Vectors Shocked Into Bacteria
Next, we bring together the vectors and bacteria cells and expose the mixture to a brief electrical shock. The shock forces some of the vectors into the cells of the bacteria.

If a cell takes in more than one vector (they almost never do), that cell will not go on to replicate.

Multiplication

Let Cells Multiply
The cells are spread out thinly on a growth medium and allowed to multiply. The cells are far enough away from one another to establish their own distinct colonies.

As the cells multiply and duplicate their own chromosomes, they duplicate the vectors as well. Each bacterial daughter cell ends up with a copy of the vector, which includes the human DNA fragment. Each colony on the plate will contain copies of a different piece of cloned DNA.

Pick a Colony
When a colony has grown to about the size of a period in a book, it will contain a million cells. At this point we place the colony in a liquid culture, where it multiplies further.

Soon there are billions of copies of the original cell. Each cell contains a copy of the vector and within that vector the original human DNA fragment.

Recover

Recover Cloned DNA
We then expose all of the cells to a detergent, which ruptures the cells' walls, and to sodium hydroxide, which causes the bacteria's chromosomes to deteriorate and fall away from the vectors.

The circular vectors are affected by this treatment, too, but they quickly recover.

Repeat

Repeat
So we now have billions of copies of human DNA (still attached to the vector), all derived from a single DNA fragment.

Each copy of this human DNA is 150,000 base pairs long, which is still too long for determining the sequence of its code. To get a batch of smaller fragments, we repeat the entire process just described. We end up with billions of copies of a fragment 2,000 to 4,000 base pairs long.

Continue: Part III: Preparing DNA for Detection

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