Introduction To Molecular Biology

.Prasanth H

Company Name
 Molecular Biology
 Molecular biology; the study of gene structure and functions at
the molecular level to understand the molecular basis of
hereditary, genetic variation, and the expression patterns of
genes.
 The Molecular biology field overlaps with other areas,
particularly genetics and biochemistry.
 The genome of an organism is the totality of genetic
information and is encoded in the DNA (or, for some viruses,
RNA).
 The genome database is organized in six major organism
groups: eukaryotes, bacteria, archaea, viruses, viroids and
plasmids.
 All living things are grouped into three domain: eukaryotes;
prokaryotes and archaea.
 The DNA
 Deoxyribonucleic Acid (DNA), the genetic material of all cellular
organisms and most viruses, the gigantic molecule which is
used to encode genetic information for all life on Earth.
 The Chromosome, the storage place for all genetic
information, the number of chromosomes varies from one
species to another.
 In normal human cell DNA contained in the nucleus, arranged
in 23 pairs of chromosomes ; 22 pairs of chromosomes
(autosomes); the 23 chromosome pair determines the sex of
individual and is composed of either two (x) chromosomes
(female) or an (x) and (y) chromosome (male).
 The gene, the basic units of inheritance; it is a segment within
a very long strand of DNA with specific instruction for the
production of one specific protein. Genes located on
chromosome on it's place or locus.
 The Cell
 The cell is the smallest living unit, the basic structural and
functional unit of all living things. Some organisms, such as
most bacteria, are unicellular consist of a single cell. Other
organisms, such as humans, are multicellular.
 Cells are stacked together to make up structures, tissues and
organs. Most cells have got the same information and
resources and the same basic material. Cells can take many
shapes depending on their function.
 Function of cells
 *Secretion (Produce enzymes).
 *Store sugars or fat.
 *Brain cells for memory and intelligence.
 *Muscle cells to contract.
 *Skin cell to perform a protective coating.
 *Defense, such as white blood cells.
 Eukaryotic Cell
Eukaryotes are generally more advanced than prokaryotes.

Eukaryotic cells are found in animals; plants; fungi and protists

cell.
 Cell with a true nucleus, where the genetic material is
surrounded by a membrane;
 Eukaryotic genome is more complex than that of prokaryotes
and distributed among multiple chromosomes;
 Eukaryotic DNA is linear;
 Eukaryotic DNA is complexed with proteins called histones;
 Numerous membrane-bound organelles;
 Complex internal structure;
 Cell division by mitosis.
 Eukaryotic Cell Cycle: defined as the sequence of events that
occurs during the lifetime of a cell and is traditionally divided
into four phases:

G1 = Growth and preparation of the chromosomes for replication

S = Synthesis of DNA
G2 = Preparation for mitosis
M = Mitosis
 Prokaryotic Cell
 Unicellular organisms, found in all environments. These
include bacteria and archaea.
 Without a nucleus; no nuclear membrane (genetic material
dispersed throughout cytoplasm;
 No membrane-bound organelles;
 Cell contains only one circular DNA molecule contained in the
cytoplasm;
 DNA is naked (no histone);
 Simple internal structure; and
 Cell division by simple binary fission.

 Archaea are prokaryotes; organisms without nucleus but

some aspect of their molecular biology are more similar to
those of eukaryotes.
Eukaryotic Cell
Central Dogma of Molecular
Biology
 General Structure of Nucleic
Acid
 DNA and RNA are long chain polymers of small compound called
nucleotides. Each nucleotide is composed of a base; sugar (ribose in
RNA or deoxyribose in DNA) and a phosphate group. The phosphate
joins the sugars in a DNA or RNA chain through their 5` and 3`
hydroxyl group by phosphodiester bonds.

 The structure of DNA was described by British Scientists Watson and

Crick as long double helix shaped with its sugar phosphate backbone
on the outside and its bases on inside; the two strand of helix run in
opposite direction and are anti-parallel to each other. The DNA double
helix is stabilized by hydrogen bonds between the bases.

 This structure explains how genes engage in replication, carrying

information and acquiring mutation.

 The G+C content of a natural DNA can vary from 22-73% and this can
have a strong effect on the physical properties of DNA, particularly its
melting temperature.
 There are four different types of nucleotides found in DNA,
differing only in the nitrogenous base: A is for adenine; G is for
guanine; C is for cytosine and T is for thymine.

 These bases are classified based on their chemical structures

into two groups: adenine and guanine are double ringed
structure termed purine , thymine and cytosine are single ring
structures termed pyrimidine.

 The bases pair in a specific way: Adenine A with thymine T

(two hydrogen bonds) and guanine G with cytosine C (three
hydrogen bonds).

 Within the structure of DNA, the number of thymine is always

equal to the number of adenine and the number of cytosine is
always equal to guanine.

 In contrast to DNA; RNA is a single stranded, the pyrimidine

base uracil (U) replaces thymine and ribose sugar replaces
deoxyribose.
 Genomic DNA Organization
 Eukaryotic genes: DNA molecules complexed with other
proteins especially basic proteins called histones, to form a
substance known as chromatin. A human cell contains about 2
meters of DNA. DNA in body could stretch to the sun and back
almost 100 times. So it is tightly packed.

 Eukaryotic chromatin is folded in several ways. The first order

of folding involves structures called nucleosomes, which have
a core of histones, around which the DNA winds ( four pairs of
core histones H2A, H2B,H3 and H4 in a wedge shaped disc,
around it wrapped a stretch of 147 bp of DNA). The second
order of chromatin folding is the 30 nm fiber. The third order of
chromatin condensation appears to involve formation of a
radial loop structure in eukaryotic chromosomes.
Genomic DNA Organization
 DNA Replication
 DNA Replication: The DNA (all gene duplication; the transfer the
genetic information from a parent to a daughter cell ; the DNA base
sequence are precisely copied).

 Semiconservative replication describes the method by which DNA is

replicated in all known cells. This method of replication was one of
three proposed models of DNA replication:
 Conservative replication would leave the two original template DNA
strands together in a double helix and would produce a copy
composed of two new strands containing all of the new DNA base
pairs.
 Dispersive replication would produce two copies of the DNA, both
containing distinct regions of DNA composed of either both original
strands or both new strands.
 Semiconservative replication would produce two copies that each
contained one of the original strands and one entirely new strand.
 DNA Forms
•A Form
•B Form
•Z Form
 Mitochondria
 Mitochondria is a membrane enclosed organelle found in most
eukaryotic cells. These organelles range from 1-
10 micrometers (μm) in size.

 Mitochondria generate most of the cell's supply of adenosine

triphosphate (ATP).

 Mitochondria are involved in a range of other processes, such

as signaling, cellular differentiation, cell death, as well as the
control of the cell cycle and cell growth.

 Mitochondria have been implicated in several human diseases,

including mental disorders, cardiac dysfunction, and may play
a role in the aging process.

 Mitochondria has its own DNA.

 Mitochondrial DNA
 Mitochondrial DNA contains 37 genes, all of which are
essential for normal mitochondrial function.

 Thirteen of these genes provide instructions for making

enzymes involved in oxidative phosphorylation.

 Oxidative phosphorylation is a process that uses oxygen and

simple sugars to create adenosine triphosphate (ATP), the
cell's main energy source.

 The remaining genes provide instructions for making

molecules called transfer RNAs (tRNAs) and ribosomal RNAs
(rRNAs).

 Mitochondrial genes are among the estimated 20,000 to 25,000

total genes in the human genome.
 The Genetic Code
 The purine and pyrmidine bases of the DNA molecule are the
letters or alphabet of the genetic code. All information
contained in DNA represented by four letters: A,T,C,G.

 Three nucleotides of DNA (1st, 2nd and 3rd) form triplet

codons, there are 64 possible codons, most amino acids have
more than one possible codon. Out of the 64 possible 3-base
codons, 61 specify amino acids; the other three are stop
signals (UAG, UAA, or UGA).

 The sequence of codons in the mRNA defines the primary

structure of the final protein.
http://www.accessexcellence.org/RC/VL/GG/genetic.php
Series of codons in part of amRNA molecule. Each codon consists of three
.nucleotides , representing a singleamino acid
 The RNA
 Three major classes of RNA: messenger (mRNA), transfer
(tRNA) and ribosomal (rRNA). Minor classes of RNA include
small nuclear RNA ; small nucleolar RNA;………..

 mRNA molecules represent transcripts of structural genes

that encode all the information necessary for the synthesis of a
single type polypeptide of protein.
 mRNA; intermediate carrier of genetic information; deliver
genetic information to the cytoplasm where protein synthesis
take place.
 The mRNA also contains regions that are not translated: in
eukaryotes this includes the 5' untranslated region, 3'
untranslated region, 5' capand and poly-A tail.
 The RNA
 All tRNAs share a common secondary structure represented by a
coverleaf. They have four- base paired stems defining three stem
loops (the D loop, anticodon loop, and T loop) and the acceptor stem
to which amino acids are added in the charging step.

 RNA molecules that carry amino acids to the growing polypeptide.

 Ribosomal RNA (rRNA) is the central component of the ribosome, the

function of the rRNA is to provide a mechanism for decoding mRNA
into amino acids and to interact with the tRNAs during translation by
providing peptidyl transferase activity.

 The concentration of purine and pyrimidine bases do not necessarily

equal one another in RNA because RNA is single stranded. However,
the single strand of RNA is capable of folding back on itself like a
hairpin and acquiring double strand structure.
 mRNA
tRNA
 The Protein
 Proteins are the basic building materials of a cell, made by cell
itself; the final product of most genes.

 Proteins are chain like polymers of a few or many thousands of

amino acids. Amino acids are represented by codons, which
are 3-nucleotide RNA sequences. Amino acids joined together
by peptide bonds (polypeptide). Proteins can be composed of
one or more polypeptide chains.

 Proteins have many functions: provide structure that help cells

integrity and shape (e.g. collagen in bone); serve as enzymes
and hormones; bind and carry substance and control of
activities of genes….
Four levels of protein structure
 Primary structure: Formed by joining the amino acid sequence
into a polypeptide.

 Secondary structure: Different conformation that can be taken

by the polypeptide: alpha helix and strands of beta sheet.

 Tertiary structure : Result from folding the secondary structure

components of the polypeptide into three-dimensional
configuration.

 Quaternary structure : complex of several protein molecules or

polypeptide chains, usually called protein subunits, which
function as part of the larger assembly or protein complex.
 Ribosomes
 Ribosomes; Factory for protein synthesis; are composed of
ribosomal RNA and ribosomal proteins (known as a
Ribonucleoproteinor RNP). They translate messenger RNA
(mRNA) to build polypeptide chains using amino acids
delivered by transfer RNA (tRNA).

 Eukaryotic ribosomes are larger. They consist of two subunits;

a 60S subunit holds (three rRNAs 5S, 5.8S, 28S and about 40
proteins) and a 40S subunit contains (an18S rRNA and about
30 proteins) , which come together to form an 80S particle
compared with prokaryotic 70S ribosome.

 Most mRNA are translated by more than one ribosome at a

time; the result, a structure in which many ribosomes translate
an mRNA in tandem, is called a polysomes.
Ribosomes

Four levels of protein structure

Polysomes
 Gene Expression
 Gene expression process by which a gene product
(an RNA or polypeptide ) is made.

 In transcription steps, RNA polymerase make a copy

of information in the gene (complementary RNA)
(mRNA) complementary to one strands of DNA.

 In translation step, ribosomes read a messenger

RNA and make protein according to its instruction.
Thus any change in gene sequence may lead to
change in the protein product.
 Types of control in eukaryotes
 Transcriptional, prevent transcription, prevent mRNA from
being synthesized.

 Posttranscriptional, control mRNA after it has been produced.

 Translational, prevent translation; involve protein factors

needed for translation.

 Posttranslational, after the protein has been produced.

 Mutation
 Mutation include both gross alteration of chromosome and more
subtle alteration to specific gene sequence.

 Gross chromosomal aberrations include: large deletions; addition and

translocation (reciprocal and nonreciprocal).

 Mutation in a gene's DNA sequence can alter the amino acid

sequence of the protein encoded by the gene. Point mutations are the
result of the substitution of a single base. Frame-shift mutations occur
when the reading frame of the gene is shifted by addition or deletion
of one or more bases.

 Mutations can have harmful, beneficial, neutral, or uncertain effects on

health and may be inherited as autosomal dominant, autosomal
recessive, or X-linked traits. Mutations that cause serious disability
early in life are usually rare because of their adverse effect on life
expectancy and reproduction.
 Common Tools of Molecular Biology

 Nucleic acid fractionation

 Polymerase chain reaction
 Probes, Hybridization
 Vector, Molecular cloning
 Nucleic acid enzymes
 Microarray
 DNA sequencing
 Electrophoretic separation of nucleic acid
 Detection of genes:
*DNA: Southern blotting; inSitu hybridization; FISH Technique
*RNA: Northern blotting
*Protei n: Western blotting, immunohistochemistry
 Human Genome Project

Goals
 Identify all the approximately 20,000-25,000 genes in human
DNA,
 Determine the sequences of the 3 billion chemical base pairs
that make up human DNA, store this information in databases,
 Improve tools for data analysis, transfer related technologies to
the private sector, and
 Address the ethical, legal, and social issues (ELSI) that may
arise from the project.
Molecular biology is facilitating research in many field including
biochemistry, microbiology, immunology and genetics,…………………
…

Various methods in molecular biology diagnose the different human

diseases; diagnosis of an infectious agent, in malignancy, the
presence of the genetic disease and in transplantation, paternity and
forensic analysis.

The most recent applied technologies, genetic engineering, DNA finger-

printing in the social and forensic science, pre and postnatal
diagnosis of inherited disease, gene therapy and drug Design.

Molecular biology allows the laboratory to be predictive in nature, it gives

information that the patients may be at risk for disease (future).
Glossary
 Alleles are forms of the same gene with small differences in their sequence of DNA bases.
 Exon (Coding DNA): A gene sequence contains protein coding information.
 Introns (intervening sequence) (A noncoding DNA sequence ): Intervening stretches of DNA that separate exons.
 Primary transcript: The initial production of gene transcription in the nucleus; an RNA containing copies of all exons and introns.
 RNA gene or non-coding RNA gene: RNA molecule that is not translated into a protein. Noncoding RNA genes produce transcripts that
exert their function without ever producing proteins. Non-coding RNA genes include transfer RNA (tRNA) and ribosomal RNA (rRNA),
small RNAs such as snoRNAs, microRNAs, siRNAsand piRNAs and lastly long ncRNAs.
 Enhancers and silencers: are DNA elements that stimulate or depress the transcription of associated genes; they rely on tissue specific
binding proteins for their activities; sometimes a DNA elements can act either as an enhancer or silencer depending on what is bound to
it.
 Activators: Additional gene-specific transcription factors that can bind to enhancer and help in transcription activation.
 Open reading frame (ORF): A reading frame that is uninterrupted by translation stop codon (reading frame that contains a start codon and
the subsequent translated region, but no stop codon).
 Directionality: in molecular biology, refers to the end-to-end chemical orientation of a single strand of nucleic acid. The chemical
convention of naming carbon atoms in the nucleotide sugar-ring numerically gives rise to a 5' end and a 3' end ( "five prime end" and
"three prime end"). The relative positions of structures along a strand of nucleic acid, including genes, transcription factors, and
polymerases are usually noted as being either upstream (towards the 5' end) or downstream (towards the 3' end).
 3' flanking region: Present adjacent to 3' end of the gene; often contain sequences which affect the formation of the 3` end of the
message and may contain enhancers or protein binding sites.
 5' flanking region: A region adjacent to 5' end of the gene. It is not transcribed into RNA; it contains the promoter. May contain enhancers
or other protein binding sites.
 3' untranslated region: The three prime untranslated region (3' UTR) is a particular section of messenger RNA (mRNA). It follows the
coding region. It is a region of the DNA which is transcribed into mRNA and becomes the 3' end or the message, Several regulatory
sequences are found in the 3' UTR. The 3' untranslated region may affect the translation efficiency of the mRNA or the stability of the
mRNA. It also has sequences which are required for the addition of the poly(A) tail to the message (including one known as the
"hexanucleotide", AAUAAA).
 5' untranslated region: The five prime untranslated region (5' UTR), also known as the leader sequence, is a particular section of
messenger RNA (mRNA) and the DNA that codes for it. It is a region of a gene which is transcribed into mRNA. It starts at the site (where
transcription begins) and ends just before the start codon (usually AUG) of the coding region. It usually contains a ribosome binding site
(RBS), in bacteria also known as the Shine Dalgarno sequence (AGGAGGU). In prokaryotic mRNA the 5' UTR is normally short. Some
viruses and cellular genes have unusual long structured 5' UTRs which may have roles in gene expression. Several regulatory sequences
may be found in the 5' UTR.
 Reverse Transcription: Some viruses (such as HIV, the cause of AIDS), have the ability to transcribe RNA into DNA.
References and Online Further Reading

 Robert F. Weaver. Molecular Biology. Fourth Edition. McGraw-Hill International Edition. ISBN 978-0-07-110216-2
 Innis,David H. Gelfand,John J. Sninsky PCR Applications: Protocols for Functional Genomics: ISBN:0123721865
 Daniel H. Farkas. DNA Simplified: The Hitchhiker's Guide to DNA. Washington, DC: AACC Press, 1996, ISBN 0-915274-84-1.
 William B. Coleman,Gregory J. Tsongalis: Molecular Diagnostics: For the Clinical Laboratorian: ISBN 1588293564...
 Robert F. Mueller,Ian D. Young. Emery's Elements of Medical Genetics: ISBN. 044307125X
 Daniel P. Stites,Abba T. Terr. Basic Human Immunology: ISBN. 0838505430
 Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter. Molecular Biology of the cell. ISBN.
9780815341055
 http://www.pubmedcentral.nih.gov/
 http://www.biomedcentral.com/1471-2105/2/8/abstract. Elena Rivas and Sean R Eddy Noncoding RNA gene detection using comparative
sequence analysis
BMC Bioinformatics 2001, 2:8doi:10.1186/1471-2105-2-8
 www.medscape.com
 http://www.medterms.com/script/main/art.asp?articlekey=4026
 www.emedicine.com
 www.ebi.ac.uk/2can good introduction to bioinformatics and molecular biology
 http://www.genomicglossaries.com/
 http://www.gene.ucl.ac.uk/nomenclature/guidelines.html defines the nomenclature for human genes
 http://www.accessexcellence.org
 http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Codons.html
 http://www.web-books.com/MoBio/
 http://www.expasy.org
 http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookPROTSYn.html
 Cell & Molecular Biology online: http://www.cellbio.com/recommend.html
 http://www.ornl.gov/sci/techresources/Human_Genome/glossary/glossary.shtml%20
 http://www.genome.gov/10000715
 http://www.ncbi.nlm.nih.gov/About/primer/mapping.html
 http://www.lilly.com/research/discovering/targets.html
 http://www.informatics.jax.org/expression.shtml
 www.wikipdia.com
 http://www.biology.arizona.edu/cell_bio/tutorials/pev/page2.html
 http://www.genome.ou.edu/protocol_book/protocol_index.html
Thank
you