CPP 501 - Introduction To Molecular Genetics
CPP 501 - Introduction To Molecular Genetics
genetics
• The transmission of traits was due to a substance called Deoxyribonucleic Acid (DNA).
• It was determined later that the method by which DNA works to carry and transmit
information about the organism was through its sequence of nitrogenous bases
(Guanine, Cytosine, Adenine & Thymine).
• The structure of DNA is a double helix where Adenine is complement to Thymine and
Guanine is complement to Cytosine.
• The sequence of these bases along a strand of DNA determines the function of the DNA
at the location (called a locus).
• The primary purpose of DNA is therefore to provide the genetic code for proteins to be made, which
is the manifestation of the genetic code.
• The 1st step in this process is the transcription of DNA, where a DNA polymerase protein binds to
the DNA and transcribes a copy of the template DNA called mRNA.
• After the mRNA has left the nucleus, the sequence of the mRNA strand gets interpreted to form
proteins, biological compounds that act as the building blocks of most cellular function.
• Proteins are made up of structures called Amino Acids bonded together in a sequence specified by
the mRNA strand.
• Every 3 nucleotides (nitrogenous base plus the sugar backbone of nucleic acid) represent a codon
and depending on the codon, the ribosome will add a specific Amino-acid to the protein being
synthesized.
• These proteins ultimately mediate most cellular functions and determine almost all of the features
that are expressed in organisms.
Summary
Molecular Genetics is the study of agents that pass information from
generation to generation. These molecules (genes) are long polymers of DNA.
Just four chemical building blocks:
• Guanine (G)
• Adenine (A)
• Thymine (T)
• Cytosine (C)
Are placed in a unique order to code for all of the genes in a living organism
Properties of a Genetic Material
• A genetic material must be able to control the phenotype of a cell or
organisms (i:e to direct protein synthesis).
• Bases from the 2 strands form hydrogen bonds with each other with the
restriction that only A and T or G and C can pair.
• DNAs with higher G-C contents have high melting point or denaturation.
• G-C base pairs have three hydrogen bonds versus only 2 in an A-T base
pair.
DNA Replication
• DNA replicates by unwinding of the double helix, with each strand subsequently acting as a
template for a new strand.
• This works because of complementarity – Only A-T, T-A, G-C or C-G base pairs form stable
hydrogen bonds within the structural constraints of the model.
• Replication proceeds in small segments working backward from the Y-junction on the 5’ – 3’
template strand.
• Polymerase III is the active replicating enzyme and polymerase I is involved DNA repair.
• Molecular Genetics is a genetics sub-discipline including areas
such as DNA structure, RNA structure, gene expression, gene
mutation and gene therapy.
• These genes are units of hereditary that are stored inside cell
and passed down through generations
Chemical Structure of DNA
• DNA is a long molecule with a double helix structure which is
sometimes compared to a spiral staircase.
• These bases are paired along the helix to form the stairs. They
are
• Adenine (A)
• Thymine (T)
• Cytosine (C)
• Guanine (G)
Chemical Structure of RNA and Gene Expression
• Ribonucleic Acid (RNA) is similar to DNA
• On paper, a DNA strand can be coded from RNA simply by keeping this difference in chemical
bases in mind, e.g the RNA sequence AUUGGC corresponds to the DNA sequence TAACCG.
• Inside the cell’s nucleus messenger RNA (mRNA) takes a copy of DNA.
• It then leaves the nucleus where organelles called ribosomes need this information to make
proteins.
• Gene Expression is used to describe the process of transcription and translation that leads to the
production of a protein.
Molecular Genetics and Gene Mutation
• Many of the enzymes that copy DNA, make RNA from DNA, and
synthesize proteins from RNA template were first characterized in bacteria.
Obtaining DNA for Laboratory Analysis
• One way of obtaining many copies of DNA is to isolate DNA from
millions of cells grown artificially in the laboratory.
• It involves separating DNA from other cellular components such as proteins, RNA and
lipids.
• The cell used to obtain and isolate the DNA could come directly from tissue or could be
cultured laboratory cell lines.
• DNA is isolated by placing the cells in a tube containing a special solution called a
“cocktail” and mechanically or chemically breaking them open.
• This causes the cell to release its contents into the cocktail containing enzymes,
chemicals and salts.
• Enzymes are used to chew up the proteins, chemicals to destroy any RNA present and
salts to help pull the DNA out of solution.
DNA Isolation Cont’d
• At this point, the DNA will exist in a long strands that form a mucus-like glob
within the solution.
• The solution is then poured off, and the DNA is dissolved or re-suspended in a
second solution that will make it easy to work with in subsequent procedures.
• To make a clone, a target DNA sequence is inserted into what is called a cloning vector.
• A cloning vector is a DNA molecule originating from a virus, plasmid, or the cell of a
higher organism into which another DNA fragment of appropriate size can be integrated
without interfering with the vector; capacity for self-replication.
• The target and vector DNA fragments are then ligated or joined together, to create what
is called a recombinant DNA molecule.
• Recombinant DNA molecules are usually introduced into Escherichia coli (E. Coli) – a
common laboratory strain of a bacterium – by transformation (the natural DNA Uptake
mechanism possessed by bacteria)
2. Polymerase Chain Reaction (PCR)
• The Polymerase Chain Reaction is simple technique that results in the exponential amplification of
almost any region of a selected DNA molecule.
3) Primers, single stranded DNAs between 20 & 50 nucleotides long that are complimentary to a short
region on either side of the template DNA
4) Taq polymerase, a heat stable enzyme that drives or catalyzes the synthesis of new DNA
• The PCR is carried out by mixing together in a small test tube the template DNA , DNA
nucleotides, primers and Taq polymerase.
• The primers must anneal or pair to the template DNA on either side of the region that is
to be amplified or copied.
• This means that the DNA sequences of these borders must be known so that the
appropriate primers can be made.
• These nucleotides serve to initiate the synthesis of the new complementary strand of
DNA.
• Because Taq polymerase, a form of DNA polymerase that catalyzes the synthesis of new
DNA in incredibly heat stable, the reaction mixture can be heated to approximately 90 0C
without destroying the molecules, enzymatic activity.
• At this temperature, the newly created DNA strands detach from the template DNA
• The reaction mixture is then cooled again allowing more primers to anneal to
the template DNA and also to the newly created DNA.
• The Taq polymerase can now carry out a second cycle of DNA synthesis.
• This process of heating, cooling and heating is repeated over and over.
Because each cycle doubles the amount of template DNA in the previous
cycle, one template DNA molecule rapidly becomes hundreds of thousands of
molecules in just a couple of hours
• The chain termination method is the method commonly used today. This method is based on
principle that DNA molecules that differ in length by just a single nucleotide can be separated
from one another using polyacrylamide gel electrophoresis.
• The DNA to be sequenced called the template DNA is 1st prepared as a single-stranded DNA.
• Next a short oligonucleotide is annealed or joined to the same position on each template
strand.
• The oligonucleotide acts as a primer for the synthesis of a new DNA strand that will be
complementary to the template DNA.
• This technique requires that 4 nucleotide specific reactions - one each for G, A, C and T be
performed on 4 identical samples of DNA