DNA

As we mentioned in the first section the basic unit of life in most organism is called DNA (Deoxyribo nucleic Acid). The DNA represents the genetic material of an organism. RNA (Ribo Nucleic Acid) at one point might have served as DNA but due to lack of stability of RNA evolution likely created DNA (much more stable) to be the repository of genetic information.

1. Both DNA and RNA are composed of nucleotides which consist of a sugar, phosphate group and a base (purine and pyrimidine)

2. DNA exists in a double helical structure which is anti-parallel which is important for DNA replication

3. The nucleotides are the monomers of nucleic acids (DNA and RNA) which serve a variety of functions within a cell. They serve as holders of genetic material, energy coins (ATP, GTP), and as cofactors in certain reactions.

Nucleotides consists of three things:

1. nitrogeneous base (purine or pyrimidine)

a. Purine

1. Adenine (found in DNA and RNA)
2. Guanine (found in DNA and RNA)

b. Pyrimidines

1. Cytosine (found in DNA and RNA)
2. Thymine (found in DNA only)
3. Uracil (found in RNA only)

c. DNA bases are such that one purine can only form a complimentary pair with one other pyrimidine and vice-versa. Any other incorrect or mismatch base pairs is responsible for mutations which are potentially deleterious to the body.

2. pentose sugar (ribose)

A. deoxyribose in DNA

1. In DNA the 2' OH of ribose is replaced by a hydrogen atom

B. ribose in RNA

1. In RNA the there is a hydroxyl group on both the 2' and 3' position. This contributes to RNA's inherent poor half-life

C. The ribose ring is not planar it is puckered with either C-2' or C-3' out of the plane of the moelcule.

D. The nitrogeneous base is attached covalently to C-1' position of ribose via N-glycosidic bond to either the N1 position of pyrimidine or N9 position of purines. Make sure you understand the numbering in the pictures

3. phosphate group (which links one sugar to the next leading to the formation of the DNA polymer)

1. The phosphate groups is esterified to C-5' of ribose. Free nucleotides exist as either NTP, NDP, NMP (Nucleotide tri, di, or mono phosphate)

DNA

• Nucleotides are linked together via a covalent bond called a phosphodiester bond. The 5-OH of one sugar is linked to the 3' OH of the subsequent sugar. As a result each chain has a 5' end and a 3' end. Which gives DNA its directionality.
• The DNA backbone which contains ribose and phosphate groups is highly hydrophilic.
• As I mentioned earlier DNA is many times more stable than RNA due to the lack of 2' hydroxyl group. The 2' hydroxyl in RNA undergoes base catalyzed hydrolysis and hence leads to the short half life of DNA. Over the years evolution switched how genetic material was stored from DNA to RNA.
• The nitrogenous bases are both hydrophobic and planar. Due to the planaraity adjacent bases stack on each other. The bases contain ring nitrogens, exocyclic nitrogens, and carbonyl all can participate in hydrogen bonding. The complimentary which maximizes the hydrogen bonding between adjacent bases on anti-parallel strands (between donor and acceptor) led to the idea that Adenine pairs with Thymine (or Uracil) and Guanine pairs with Cytosine. Adenine and Thymine pair with each other due to the formation of two hydrogen bonds while Guanine and Cytosine pair with each other by the formation of three hydrogen bonds.

DNA as a mechanism for storage of genetic information

• Structure of DNA determined in 1953 (Watson and Crick) which allowed you to understand how information is passed from one generation to the next.
• The structure determined by crystal structure showed that DNA is anti-parallel double helix. The strands are held together by complimentary base pairing. The bases are found on the inside while the backbone is hydrophilic and found on the outside. The bases are stacked off center which leads to a ladder like structure that goes up into the helix. There are around 10.5 base pairs per turn of the helix. The helix is RIGHT HANDED which creates two grooves: the major and minor groove. The major and minor groove serve as areas of recognition due to particular consensus sequences that can be bound by protein or other molecules to regulate gene expression.
• The complimentary base pairing allowed for further elucidation of how DNA is replicated and passed on: Each strand contains enough information to serve as a template to replicate. (The information is presented twice just to prevent the number of errors that could be formed).
• Non-enzymatic reactions can lead to mutations. These include deamination of cytosine, UV light which forms thymine dimers (stay away from the sun), Depurination, and others.

Methods: DNA sequencing

It is easy and routine to determine the sequence of a stretch of DNA hundreds of nucleotides long.

History of DNA sequencing:

1. Maxim and Gilbert: 1977 It uses differential chemical reactivity of the baes with various 'reagents' to selectively induce strand cleavage. These fragments were then run on a PAGE Gel. The lanes were usually A A+T G C+G. These special reagens were such that they cleaved before a adenine, before either a adenine or a thymine, before a guanine or before a cytosine or guanine. As youc an see this is quite tedious and requires large amounts of DNA.

2. Sanger Sequencing- His method uses an enzymatic reaction to sequence DNA. An oligonucleotide primer is extended by a DNA polymerase in the presence of each of the four nucleoside triphosphates and a low concentration of one dideoxy nucleoside triphosphate. The polymerase produces a new chain complementary to one strand of the DNA, but each time the polymerase reaches a base complementary to the one for which a dideoxy nucleotide is present, there is a chance that a dideoxy nucleotide will be incorporated. Although it is readily incorporated, the dideoxy nucleotide is a chain terminator (it does not have a 3’OH, which is required for subsequent extension of the chain). Four reactions are performed, one with each dideoxy nucleotide and the results shown on a denaturing PAGE GEL.

3. Automation- Currently automated DNA sequencing is used where fluorescent dyes are used to generate four distinct signals. Instead of four separate reactions like the Sanger, all the reactions are run at the same time, this is rather quick and able to sequence a 100-500 nucleotide sequence in a few minutes.