it is important to know the order of nucleotides in a strand of dna the process of dna sequencing...
Post on 22-Dec-2015
228 Views
Preview:
TRANSCRIPT
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
top related