14.2 DNA Structure and Sequencing

You can see how science builds upon previous discoveries after reading the past few pages.

The building blocks are called nucleotides.

A nitrogenous base, a 5-carbon sugar, and a phosphate group are important components of the nucleotide.
Depending on the nitrogenous base, the nucleotide is named.
The nitrogenous base can be either a purine or apyrimidine.

The purines have a double ring structure with a six-membered ring fused to a five-membered ring.

Pyrimidines have a single six-membered ring structure.

The five bases are shown in the images above.

The purines have a double ring structure with a six-membered ring fused to a five-membered ring.

Pyrimidines have a single six-membered ring structure.

The sugar is found in the body.

The five-carbon sugar's carbon atoms are numbered 1', 2', 3', 4', and 5'.
The 5' carbon of the sugar is connected to the 5' OH group by the formation of an ester linkage between phosphoric acid and the 5' OH group.
The 3' carbon of the sugar is attached to the OH group.
The 2' carbon of the sugar ribose has a group in it.
The base is attached to the sugar.

The bonds are produced by the combination of the nucleotides and each other.

The 5' carbon of the sugar of one nucleotide is attached to the 3' carbon of the sugar of the next nucleotide in order to form a second ester linkage.
One end of a polynucleotide has a free 5'phosphate and the other has a free 3'-OH.
The 5' and 3' ends of the chain are called the 5' and 3' ends.

The structure of DNA was determined by Francis Crick and James Watson in the 1950s.

Other scientists were exploring this field as well.

Franklin was using X-ray methods to understand the structure of DNA.

Crick studied X-ray diffraction and Franklin's data in order to piece together the puzzle of the DNA molecule.
James, Francis, and Maurice Wilkins received the prize in 1962.
Unfortunately, by Franklin's death, the prizes aren't awarded posthumously.

Our present day understanding of DNA can be traced back to the work of James Watson, Francis Crick, and Maclyn McCarty.

The X-ray pattern of DNA was discovered by scientist Rosalind Franklin.

Chargaff's Rules suggest that base pairs are made between a purine and pyrimidine on opposite strands.

Adenine and thymine are two of the complimentary base pairs.
The base pairs are stable by hydrogen bonds.
The 3' end of one strand faces the 5' end of the other strand in nature.
The nitrogenous bases are stacked inside like the rungs of a ladder, whereas the sugar andphosphates form the backbone of the structure.
Each base pair is separated from the next by a distance of less than a millimeter.
10 base pairs are present per turn of the helix.
The diameter of the double-helix is 2 nm.
The diameter can only be explained by the pairs of purine and pyrimidine and the antiparallel orientation of the two strands of DNA.
The grooves are formed by the twisting of the strands around each other.

A double helix structure and hydrogen bonds are found in DNA.

The major and minor grooves are where the binding sites for the DNA binding proteins are located.

The process of reading the sequence of DNA was long and expensive before the 1990s.

The problem was compounded by using radiolabeled nucleotides.

The method used for the human genome sequencing project was developed by Fred Sanger.

There is a link to learning to watch a video about the sequence-reading technique that resulted from Sanger's work.

The dideoxy chain terminated method is known as the sequencing method.

The method is based on chain terminators.
The chain cannot be extended further if a free 3' OH group is not available.
It is possible to create different sized DNA fragments by using a ratio of de and dide.

Frederick Sanger's dideoxy chain terminated method uses dye-labeled dideoxynucleotides to generate fragments that end at different points.

The DNA is separated by capillary electrophoresis on the basis of size, and from the order of fragments formed, the sequence can be read.
The laser scanned DNA sequence is shown on apherogram.

The sample is separated into two strands by heating it to high temperatures.

A primer, DNA polymerase, and all four nucleoside triphosphates (A, T, G, and C) are added to the DNA in four tubes.
There are limited quantities of one of the four dideoxynucleoside triphosphates added to each tube.
The tubes are labeled as A, T, G, and C. Each of the four dideoxynucleotides has a different fluorescent label.
After a fluorescent dideoxy nucleotide is incorporated, there is no further chain elongation.
electrophoresis is performed after the reaction is over.
There is a difference in length of a base.
The sequence is read from a laser scanning device.
In 1980, he received a Nobel Prize for his work on genetics.

A race to sequence human genomes has led to a link to learning.

You can learn more by watching the animation here.

The gel is usually made of a chemical called agarose that is high in galactose.

The Agarose powder is heated.
The gel solution is poured into a casting tray after cooling.
The electric current is applied after the gel has solidified.
The DNA has a negative charge and moves to the positive side.
The electric current can be applied for enough time to allow the DNA to be separated according to size, and the heavier fragments will be closest to the well.
The gel is stained with a DNA-specific dye after it is separated.

The size of the DNA can be separated using gel electrophoresis.

The first draft of the Neanderthal genome was published.

They were known to have lived in Europe and Western Asia tens of thousands of years ago.
Green's team studied fossil remains from all over the world.
Because of the fragile nature of the bones, very sophisticated means of sample preparation were used.

The scientists were able to sequence four billion base pairs.

The Neanderthal sequence was compared with the present-day humans from all over the world.
The researchers found that the Neanderthal genome had more similarity to people living outside of Africa than to people in Africa.
Current theories suggest that all humans are descended from a small population in Africa, but the data from the Neanderthal genome suggest some interbreeding between Neanderthals and early modern humans.

Green and his colleagues discovered that people in Europe and Asia have the same genes as Neanderthals.

Neanderthals are as closely related to people from Australia as to those from China or France.
Neanderthal fossils have only been found in Europe and West Asia.
Neanderthals and modern humans are most likely to have had a genetic exchange before modern humans emerged out of Africa.

Several genes seem to have changed during the evolution of humans.

The genes are involved in metabolism and cognitive development.
One of the genes that is of particular interest is RunX2, which is different in modern day humans and Neanderthals.
Neanderthals had bell-shaped rib cage, prominent frontal bone, and dental differences.
The upper body and cranium are thought to have been affected by an evolutionary change in RUNX2.

The Neanderthal genome research is explained in a talk by Svante Paabo at the annual TED conference.

Eukaryotes have a lot of features that are simpler than prokaryotes.

The nucleus of most prokaryotes contains a single, circular chromosome.

The nucleus of a eukaryote is well-defined, whereas the nucleus of a prokaryote is not.

Both processes occur in prokaryotic cells.

If cut and stretched out, the size of the E.coli genome is approximately 1.1 million base pairs.

Supercoiling is when the DNA is twisted.

Supercoiling suggests that DNA is either "under-wound" (less than one turn of the helix per 10 base pairs) or "over-wound" (more than 1 turn per 10 base pairs) from its normal relaxed state.

Some genes are involved in the supercoiling and other genes are involved in maintaining the structure.

Eukaryotes use different packing strategies to fit their DNA inside the nucleus.

The histones are composed of two molecules of each of four different histones and are evolutionarily conserved.
The histone core is wrapped around the negatively charged DNA.
The next one is linked with the help of a linker DNA.
The beads on a string structure is also known as this.
A string of nucleosomes is further compressed into a 30-nanometer fiber with the help of a fifth histone.
Metaphase chromosomes are further reduced by association with scaffolding proteins.
The chromosomes are at their most compact at the metaphase stage.

There are two distinct regions that can be distinguished by staining.

The less dense region is known as euchromatin, while the tightly packaged region is known as Heterochromatin.
Heterochromatin usually contains genes that are not expressed, and is found in the centromere and telomeres.
The genes that are transcribed are packaged around the nucleosomes.