It is important to know the order of nucleotides in a strand of DNA

The process of DNA sequencing (determining the order of nucleotides) uses processes found in vivo (inside the cell)

Recall that DNA polymerase adds complementary nucleotides to a single strand of DNA

The nucleotides are found in high concentration in the cell

Scientists attempt to mimic this process in vitro (in a lab)

A length of DNA is first heated◦ This separates the two strands

Then, a heat-resistant DNA pol is added Ordinary A, C, T and G nucleotides are

added in high concentration However, a special type of each nucleotide

is also added in low concentration Two features make them unique

1.They are fluorescently labeled2.They lack two hydroxyl groups on carbon 2 and 3

(called dideoxy)

The label allows them to be detected when ran through a gel (more on this later)

The missing hydroxyls stops DNA pol from adding any more nucleotides past that point

The addition of the modified nucleotides is always random

Eventually, a set of labeled strands of all lengths is generated◦ Each ends with a dd nucleotide (one of four

possible colours) These are ran through a gel, arranging

them in order of size

The gel is passed through a detector, determining the order of the nucelotides

Sometimes, scientists want to make copies of a strand of DNA◦ For example, a small amount of DNA left at a

crime scene This can be done in vitro through a

polymerase chain reaction Involves a three step cycle

1.Heat to separate strands2.Cooling to allow annealing of DNA primers3.Addition of nucleotides via DNA pol

http://www.youtube.com/watch?v=HMC7c2T8fVk

http://www.youtube.com/watch?v=_YgXcJ4n-kQ

Recall that restriction enzymes make “sticky ends”

This works with any organism that has DNA as its genetic material

As a result, sections of DNA from one organism can be spliced into another

Bacteria is often used since it has plasmids◦ These are small, circular strands of DNA ◦ Easy to remove

The original version is called a cloning vector

Plasmids are easily removed and reinserted We have used these cloning vectors to

engineer bacteria to produce proteins such as insulin and growth hormone

Recombinant DNA

Gene for human growth hormone

Gene for human growth hormone

Human Cell

Bacteria cell

Bacterial chromosome

Plasmid

Sticky ends

DNA recombination

Bacteria cell containing gene for human growth hormone

DNA insertion

DNA that has been modified by adding foreign segments is called recombinant

It is also possible to transform multicellular organisms such as plants and animals

To do so, microorganisms, such as bacteria and viruses, must be used

For example, a bacteria that causes tumors in plants can be altered so the tumor gene is replaced by a useful gene

However, these methods can only work if the microorganism can insert its DNA into a host

Complete plant generated from transformed cell.

Inside plant cell, Agrobacterium inserts part of its DNA into host cell chromosome.

Plant cell colonies

Transformed bacteria introduce plasmids into plant cells.

Agrobacterium tumefaciens

Cellular DNA

Gene to be transferred

Recombinant plasmid