The Central Dogma of Molecular Biology

[Pages:10]Vierstraete Andy (version 1.01) 1/02/2000

The Central Dogma of Molecular Biology

-Page 1 -

Figure 1 : The Central Dogma of molecular biology.

DNA contains the complete genetic information that defines the structure and function of an organism. Proteins are formed using the genetic code of the DNA. Three different processes are responsible for the inheritance of genetic information and for its conversion from one form to another :

1. Replication : a double stranded nucleic acid is duplicated to give identical copies. This process perpetuates the genetic information.

2. Transcription : a DNA segment that constitutes a gene is read and transcribed into a

Vierstraete Andy (version 1.01) 1/02/2000

-Page 2 -

single stranded sequence of RNA. The RNA moves from the nucleus into the cytoplasm.

3. Translation : the RNA sequence is translated into a sequence of amino acids as the protein is formed. During translation, the ribosome reads three bases (a codon) at a time from the RNA and translates them into one amino acid

In eucariotic cells, the second step (transcription) is necessary because the genetic material in the nucleus is physically separated from the site of protein synthesis in the cytoplasm in the cell. Therefore, it is not possible to translate DNA directly into protein, but an intermediary must be made to carry the information from one compartment to an other

What is DNA ?

1. Nucleotides are the building stones of DNA.

There are 4 different nucleotides : ? dATP : deoxyadenosine triphosphate ? dGTP : deoxyguanosine triphosphate ? dTTP : deoxythymidine triphosphate ? dCTP : deoxycytidine triphosphate

For convenience, these 4 nucleotides are called dNTP's (deoxynucleoside triphosphates). A nucleotide is made of three major parts : a nitrogen base, a sugar molecule and a triphosphate. Only the nitrogen base is different in the 4 nucleotides.

Vierstraete Andy (version 1.01) 1/02/2000

-Page 3 -

Figure 2 : The components of nucleotides.

2. How do the nucleotides form a DNA chain ?

DNA is formed by coupling the nucleotides between the phosphate group from a nucleotide (which is positioned on the 5th C-atom of the sugar molecule) with the hydroxyl on the 3rd Catom on the sugar molecule of the previous nucleotide. To accomplish this, a diphosphate molecule is split off (and releases energy). This means that new nucleotides are always added on the 3' side of the chain.

Vierstraete Andy (version 1.01) 1/02/2000

-Page 4 -

Figure 3 : From nucleotide to DNA.

Vierstraete Andy (version 1.01) 1/02/2000

DNA in a cell

-Page 5 -

1. Which organelles contain DNA ?

Eucariotic cells contain several organelles. The nucleus contains most of the DNA in a cell and this DNA is called the chromosomal DNA. It is separated from the rest of the cell (cytoplasm) by a double layer of membrane. The mitochondria, which have a role in the oxidative degradation of nutrient molecules, also contain DNA, called the mitochondrial DNA. Eucariotic cells that are capable of photosynthesis contain chloroplasts with chloroplast DNA.

Size of genetic material

Type of DNA chromosomal DNA

Organism mammals plants fungi animals

size in base pairs 6 x 109 2 x 108 - 2 x 1011 2 x 107 - 2 x 108 16 x 103 - 19 x 103

higher plants

150 x 103 - 250 x 104

mitochondrial DNA chloroplast DNA

fungi green alga protozoa higher plants green alga

17 x 103 - 78 x 103 16 x 103 22 x 103 - 40 x 103 120 x 103 - 200 x 103 180 x 103

To have an idea of the size of this : every million bases take up a linear space of 0,34 mm. So when you take one human cell, uncoil all the chromosomal DNA and put it on a line, you would have 204 cm of DNA (a human cell contains in total 6 x 109 nucleotide pairs). To store this information on paper, you need a few pages of A4 paper : one page stores 3100 letters on one side (font courier 12), so 6 x 109 nucleotides, printed on both sides of a sheet, would need 967.742 pages (you'll have a pile of 120,96 m paper !!!). You'd better start printing immediately...

2. There are three types of genes :

1. Protein-coding genes : these are transcribed into RNA and then translated into proteins.

2. RNA-specifying genes : these are only transcribed into RNA.

3. Regulatory genes : according to a narrow definition, these include only untranscribed sequences.

The first two types are also called 'structural genes'.

Vierstraete Andy (version 1.01) 1/02/2000

-Page 6 -

Figure 4 : The DNA in a cell.

Vierstraete Andy (version 1.01) 1/02/2000

-Page 7 -

Figure 5 shows the different parts of the genes that codes for ribosomal DNA (rDNA) (important in protein synthesis (see the Central Dogma figure)). In eucariotic cells, there are 50 ? 5000 identical copies of the genes that specify 18S (small sub unit (SSU)), 5.8S and 28S (large sub unit (LSU)) in the 10 million ribosomes. These genes are tandem wise arranged in large blocks on one or more chromosomes and are separated from each other by non transcribed spacer (NTS). These genes are transcribed into a single RNA precursor from which the mature rRNA molecules are released by cleavage. This process removes the external transcribed spacer (ETS), internal transcribed spacer 1 (ITS1) and internal transcribed spacer 2 (ITS2) out of the RNA precursor and results in 3 rRNA molecules : 18S rRNA, 5.8S rRNA and 28S rRNA.

Figure 5 : The genes that codes for ribosomal DNA.

Preparation of gDNA

In eucariotic cells, the DNA is isolated in the nucleus, mitochondria (and chloroplasts). To extract the DNA, it is necessary to remove all the barriers around it. Mostly, proteinase K is used to dissolve the cell membrane and nuclear membrane, and it dissociates the proteins from the DNA. After this step, phenol is added which results in 3 phases in the tube :

1. The aqueous phase with the DNA, can be precipitated with ethanol. 2. The interface with denatured proteins. 3. The phenol phase with the dissolved proteins and fats. When the DNA is prepared, it can be checked on gel to verify the concentration and quality (sometimes, the DNA is broken down by the enzyme DNAase when the organism isn't preserved correctly for genetic research.)

Vierstraete Andy (version 1.01) 1/02/2000

-Page 8 -

Figure 6 : Verification of the prepared DNA on gel.

Principle of the PCR

The purpose of a PCR (Polymerase Chain Reaction) is to make a huge number of copies of a gene. This is necessary to have enough starting template for sequencing.

1. The cycling reactions : There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time. 1. Denaturation at 94?C :

During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example : the extension from a previous cycle). 2. Annealing at 54?C :

The primers are jiggling around, caused by the Brownian motion. Ionic bonds are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore. 3. extension at 72?C :

This is the ideal working temperature for the polymerase. The primers, where there

................
................

In order to avoid copyright disputes, this page is only a partial summary.

Google Online Preview   Download