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CPP 501 - Introduction To Molecular Genetics

The document provides an overview of molecular genetics, focusing on the transmission of traits through DNA, its structure, and the processes of transcription and translation that lead to protein synthesis. It discusses the methods for obtaining and analyzing DNA, including techniques like PCR and RFLP analysis, as well as the importance of gene mutations and gene therapy. Additionally, it outlines the chemical structure of DNA and RNA, emphasizing their roles in genetic expression and inheritance.

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0% found this document useful (0 votes)
18 views24 pages

CPP 501 - Introduction To Molecular Genetics

The document provides an overview of molecular genetics, focusing on the transmission of traits through DNA, its structure, and the processes of transcription and translation that lead to protein synthesis. It discusses the methods for obtaining and analyzing DNA, including techniques like PCR and RFLP analysis, as well as the importance of gene mutations and gene therapy. Additionally, it outlines the chemical structure of DNA and RNA, emphasizing their roles in genetic expression and inheritance.

Uploaded by

ridwankorede246
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
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Introduction to molecular

genetics

Oluwranti Abimbola Ph. D,


Department of Crop Production and Protection,
Faculty of agriculture,
Obafemi Awolowo University, Ile-Ife,
Nigeria
Introduction
• Traits are passed from one generation to the next by some mechanism.

• The field dedicated to studying this transmission of traits is known as 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.

• This process is called Translation, and occurs at proteins called ribosomes.

• 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).

• It must be able to replicate

• It must be located on the chromosomes

• DNA was observed to be the genetic material or RNA in the absence of


DNA
Structure of the DNA
• DNA was observed to be a helix of specific dimensions.

• DNA is made up of 2 strands running in opposite directions with sugar-


phosphate backbones and bases facing inward.

• 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.

• This model of replication is semiconservative.

• DNA polymerase enzymes add nucleotides only in the 5’ – 3’ direction.

• 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.

• It refers to physical and chemical properties of genes

• 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.

• On each strand, there are structural units called nucleotides


which include chemicals called bases.

• 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

• RNA is a one-stranded molecule and is made of different sugars.

• It also has the chemical base uracil in place of thymine.

• 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

• Another area of interest to molecular genetics is how gene


mutates. Gene mutations are changes in an organism’s DNA
Molecular Genetics and Gene Therapy

• Gene therapy also comes under the umbrella of


molecular genetics and is concerned with
inserting new genes into organisms.
• Molecular genetics is the study of the agents that pass
information from generation to generation.

• These molecules; genes, are long polymers of deoxyribonucleic


acid or DNA .

• Just 4 chemical building blocks


• Guanine (G)
• Adenine (A)
• Thymine (T)
• Cytosine (c)
Are placed in unique order to code for all the genes in all living
organisms
Laboratory Tools and Techniques of Molecular Genetics
• The methods used by molecular geneticist to obtain and study DNA have
been developed through keen observation and adaptation of the chemical
reactions and biological processes that occur naturally in all cells.

• 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.

• Another method called cloning, uses DNA manipulation procedures to


produce multiple copies of a single gene or segment of DNA.

• The Polymerase Chain Reaction (PCR) is the 3rd method whereby a


specific sequence within a double–stranded DNA is copied, or
amplified.

• PCR amplification has become an indispensable tool in a great variety


of application
DNA Isolation
• DNA isolation refers to the process of extracting DNA from a cell in a relatively pure form.

• 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 DNA is then harvested by spinning the tube in a machine called a


centrifuge.

• During spinning, the DNA collects in the bottom of the tube.

• 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.

• The result is a concentrated DNA sample containing many thousands of


copies of each gene.
Methods for Amplifying DNA
1. Cloning DNA in Bacteria
• Cloning refers to making multiple, exact copies of a particular sequence of DNA.

• 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.

• It works in a way that is similar to DNA replication in nature.

The primary materials/reagents used in PCR are:

1) DNA nucleotides (the building blocks for the New DNA)

2) Template DNA (the DNA sequence that you want to amplify

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

PCR has many applications in biology


1) It is used in DNA mapping
2) It is used in DNA sequencing
3) It is used in molecular phylogenetics
Methods of Analyzing DNA
Once DNA has been isolated and purified, it can be further analyzed in a variety of ways,
such as to identify the presence or absence of specific sequences or to locate nucleotide
changes called mutations, within a specific sequence.
1) Autoradiography – Probing DNA
To locate a specific DNA sequence, Scientists rely on the base-pairing, or
hybridization of a short piece of DNA that is complementary to the sequence of interest.
This short, single stranded piece of DNA is called a probe and can be tagged with either
mildly radioactive nucleotides or nucleotides that are linked to a substance that emits light
when exposed to certain chemicals. The probe will be radioactively labelled. Wherever the
probe sequence complements a sequence on the membrane, it will anneal /join together to
form a region of double-stranded DNA. The membrane is then washed to remove all
unbound probe and expose to a piece of X-ray film. The detection of radioactively labeled
molecules by exposure to an X-ray sensitive photographic film is referred to as
autoradiography. Wherever the radioactively labeled probe has annealed to test DNA, a
black spot will appear on the film. This method is useful for a variety of applications
2) RFLP Analysis
Every individual has slight differences or sequence polymorphisms that make their
DNA sequence unique. These differences are often single base-pair changes that occur in
regions of DNA that do not encode a gene but which are recognized and bound by
restriction enzymes.
Restriction Enzymes are proteins that bind to a DNA molecule at a specific sequence and
make a double-stranded cut at or near that sequence.
Thus, when DNA from 2 different individuals is cut with a single restriction enzyme, DNA of
different lengths will usually be produced. This is because not all restriction sites will exist
within everyone’s DNA.
These variations in fragment length are referred to as Restriction Fragment Length
Polymorphism (RFLPs) and the pattern of fragments is unique for each person. If a
restriction polymorphism can be limited to a particular phenotype such as eye color, it is
called a restriction marker.
Restriction Markers are important because they offer a diagnostic procedure for detecting a
disease and can help a researcher isolate a gene.
DNA Sequencing
• The process of determining the order of the nucleotide bases along a DNA strand is called
sequencing .
• In 1977, 24 years after the discovery of the structure of DNA, 2 separate methods for
sequencing DNA were developed.
(i) The chain termination method
(ii) Chemical degradation

• 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

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