Download Introduction to molecular biology

Nathalie Smitz
Introduction to
molecular biology
Chromosomes, carrier of heredity
Chromosomes
 In Eukaryotic cells, chromosomes are isolated by a membrane within the
nucleus.
 The number of chromosomes can vary a lot between species but also
within it (karyotype).
Human:46 chr
Ant: 2 chr
Butterfly: 440 chr
Chromosomes
 In humans, there is two copies of each chromosome (1 coming from the
mother and 1 coming from the father) = pairs of chromosomes.
Ex. 23 pairs of chromosomes (22 pairs of nonsexual
(autosomal) chromosomes and one pair of sex chromosomes)
Chromosomes
 Each chromosome is made of a very long strand of
DNA (desoxyribonucleic acid) that is highly
compacted.
 Each DNA strand is a long double helix
containing millions of steps. The steps of the helix
consist of pairs of four types of molecules called
nucleotides.
DNA
Watson & Crick
proposed the double
helix structure in 1953.
DNA
 There are 4 nucleotide types :
 Adenine
 Guanine
 Thymine
 Cytosine
 Nucelotide complementarity:
T/A and C/G:
hydrogen bonding.
DNA
• Nucleotide succession forms genes that contains the genetic information
used to synthesize proteins. Each chromosome contains hundreds to
thousands genes (coding region) separated by non-coding regions.
Purpose of DNA
https://www.youtube.com/watch?v=zwibgNGe4aY
Transfert of the genetic
information
DNA replication
 During replication, the double strand separates itself into two.
 Replication therefore assures the transfert of the genetic information from
one cell to all its descendants during mitosis.
https://www.youtube.com/watch?v=oebogqrX5F4
Meosis
Mendel’s Principles of HeridityMendelian inheritance
Johann Gregor Mendel
 Austrian monk (1822-1884),
conducting hybridization experiments
in garden peas (Pisum sativum);
 Cultivated and tested some 5,000 pea
plants in the backyard of the church;
 Induced two generalizations which
later became known as Mendel's
Principles of Heredity or Mendelian
inheritance,
Experimental model
Feature of the garden peas (Pisum sativum) :
 Self-fertilization
 Feature observable to the naked eye: seed shape, size, color, flower color, etc.
 Developed pure lineages (= homozygote), who will give descendants that
will express unique feature
Mendel laws
First Law: Law of segregation
 When a plant with two dominant (DD) alleles is crossed with a plant
having two recessive (rr) alleles, the first generation of plants will all have
one dominant and one recessive (Dr) allele.
smooth pea (DD)
wrinkled pea (rr)
Artificial crossfecondation
Parents
Cutted stamen
before maturity
D
D
r
Dr
Dr
r
Dr
Dr
1st generation
All peas are smooth, no wrinkled pea
Mendel laws
 Autofecondation of 1st generation
individuals (phenotype: smooth)
 Wrinkled pea appears at second generation;
 Wrinkled feature is carried by the 1st
generation but not expressed ;
 In the second generation, on average one of
four plants will have two recessive alleles.
D
r
D
DD Dr
r
Dr
rr
Mendel laws
Second law: Law of independent assortment
 When parents express multiple features- ex. shape (smooth vs wrinkled)
and color (green vs yellow), the features are independently transmitted each
from another.
The hybrids of first generation F1 express
exclusively dominant features (here, yellow
and smooth)
.
At second generation, descendants will
express all possible combination between
those features.
Heredity
 Mendel wasn’t speaking about “genes” but about
transmission of some features through generations.
 In 1900, three botanists, De Vries, Correns and Von
Tschermak, performed similar experimentations with other
species model and obtained the same results.
 The word “gene” was invented to define the transmission
of features as supposed by Mendel  no link yet with
chromosomes.
Heredity
 Mendel laws were forgotten for more than 35 years, until Morgan
demonstrated that the color of fruit flies' eyes was transmitted differently
between males and females.
Morgan elaborated a theory of heredity linked to the gender: the gene
responsible of the color of the eyes in fruit flies would be located on the X
chromosome.
He therefore propose that the genetic information may be supported by the
chromosomes.
Main techniques of molecular
biology
PCR
 PCR or « polymerase chain reaction » was
invented by Kary Mullis, which obtained a
Nobel price in 1993 for this discovery.
 Important discovery because it allows to
amplify specific targeted region of the
genome.
 It evolved during the last decades but the
original principle is relatively easy.
PCR
 Exponential reaction with two principal
operators :
 The Taq Polymerase, an enzyme resistant to
high temperature, originally isolated from the
thermophile bacteria Thermus aquaticus, that can
be found nearby hot water springs.
 The dNTPs (DesoxyNucleotides-Tri-
Phosphates) are molecules containing a
nucelotide bounds to three phosphates. These are
the basic elements used by the Taq Polymerase to
synthetise complementary DNA strand.
PCR
 There are three steps :
 Denaturation : separation of the two DNA
strands through an increase of the
temperature;
 Annealing of the primers : after
temperature decrease, the specific primer
complementary to the DNA strand will
hybredize to the region to amplify;
 Elongation : the synthesis of the
complementary strand will be initiated: the Taq
polymerase will add the complementary
dNTPs, which are present in the reaction
solution.
PCR
 Different temperature for each steps:
PCR
 The PCR is an exponential reaction that uses
each produced product from one step as
matrices for the next step. This process will
produce thousands of copies of a targeted
region of the genome
 Practically, the thermocycler is the device that is used to perform
PCR by changing of temperature cyclically.
DNA sequencing
 This technic will allow to reveal which is the nucelotide succession of the
targeted genome region.
 The most used technic is the SANGER
sequencing:
 Similar to PCR, the difference is that dNTP are mixed
with ddNTP (didésoxyribonucléotides): the Taq
polymerase is blocked and the DNA strand synthesis is
stopped when a ddNTP is inserted during synthesis.
 Get an assembly of DNA amplicons of variable size,
depending on the position where the ddNTP was
inserted.
DNA sequencing
DNA sequencing
 This technic is sometimes replaced by « Next Generation Sequencing »,
which allows to obtain larger fragments of the genome, but :
 More expensive;
 Analyses complexity;
 …
Some molecular phylogenic
concepts
Molecular phylogeny
 Molecular phylogeny allows to retrace the evolutionary history of species
through the study of the evolution of mutations within specific gene(s),
using statistical methods.
 To compare obtained sequences, they must be first aligned.
Ex: MEGA, a freely accessible software, allows to do such aligments.
Molecular phylogeny
 After alignment, mutations can be identified:
 Transitions between purines or between pyrimidines
 Transversions from a purine toward a pyrimidine
 Deletions or insertions
A
C
G
T
Molecular phylogeny
 Different alignment methods exist and the choice depends of the data to
analyse and the type of analyses that will be performed.
 Some sequences are freely accessible on online repository as for example
on GENBANK.
http://www.ncbi.nlm.nih.gov/genbank/
Molecular phylogeny
 Then after, it is possible to reconstruct phylogenetic trees, associating
first the sequences that are the most similar, toward the most different
one.
 For this recontruction, we first need to identify the
evolutionay model that best fit the database. It will
evaluate:
 The evolution of one sequence in comparision to its ancestral




sequence;
The divergence between the two sequences;
The mutation frequency;
Base frequency;
Substitution rate etc
Molecular phylogeny
 The most complex evolutionary model it the GTR;
 Different results can be obtained if using different evolutionary model 
importance in choosing!
Two different phylogenetics trees
obtained with the same initial
database but using distinct
evolutionary model.
Molecular phylogeny
 When the appropriate model is selected, there are still some other
parameters to define;
 Different software are needed to buil the different trees, depending
on the methodology (Neighbour joining, bayesian analysis, maximum
likelihood, maximum parsimony)
 Different ways to recontruct a phylogenetic tree
 For more information, please refer to the ‘phylogenetic handbook’
Molecular phylogeny
 The best is to perform the tree recontructions with the different technics
and compare the obtained results between each of them.
 To have a first idea of the tree structure, you can already do one with the
MEGA software used for the alignment.
DNA Barcoding
DNA Barcoding
 DNA Barcoding is a technic that allows the identification of specimen
using a short section of DNA from a standardized region of the genome
 A good DNA barcode gene is :
 A variable sequence between specis but conserved within species, which
confers her a high discrimination power.
 A relatively short sequence sothat it may be sequenced but suffently long to
have enough informative sites.
Commonly, it is the COI gene that is used for this purpose- of about
650base pairs, at least in metazoans.
DNA Barcoding
 DNA barcoding is used for different purposes:
 Identification of cryptic species (similar morphologies  difficulties of







morphological identification)
Identification of incomplete specimens
Identification at large scale to study the biodiversity and the environmental
risks
Identification of invasive species
Identification of new species
Association of males and femelles from a same species (sexual
dimorphism)
Association of different stades of development within a same species …
Etc.
DNA Barcoding
Confirmation of morphological studies
e.g. revision of Hepsetus
DNA Barcoding
But taxonomic analyses cannot be replaced by barcoding! DNA barcoding can
help and facilitate the processus of identification and allow to discover new
species or answer to other biological questions BUT cannot replace classical
taxonomic techniques  complementarity! Ex. integrative studies.
DNA Barcoding
 Molecular barcoding is a campaign largely financed in the world, with
different organisations :
 « Barcode of Life Data Systems » (BOLD) : www.boldsystems.org
 « Consortium for Barcode of Life » (CBOL) : http://www.barcodeoflife.org/
 « European Consortium for the Barcode of Life »: http://www.ecbol.org/
 « international Barcode of Life » (iBOL): www.iBOL.org
 « The Belgian Network for DNA Barcoding » (BeBOL) :
http://bebol.myspecies.info/
 « Canadian Centre for DNA Barcoding » (CCDB) : http://www.ccdb.ca/
 « The Fish Barcode of Life Initiative » (Fish-BOL) : http://www.fishbol.org/
 ….
DNA Barcoding
 But practicaly, its not so easy…
 « Universal » primer described in the litterature are not adapted to
all organisms : additional adjustments are necessary.
Questions?