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Introduction to Genetics

ACE1013

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Genetics is the science of heredity:




How organisms pass on biological information
Study of biologically inherited traits
Study of genes

Fields of genetics

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

Transmission genetics (inheritance patterns)
Cytogenetics (chromosomes)
Molecular genetics (DNA Technology)
Population genetics (variation & evolution)
Genomics and information sciences (gene sequencing)

Selective breeding




For biggest, most useful production
o E
...
10
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of chromosomes
 Diploid: (2n) both sets
 Haploid (n) half diploid – one set
Mitosis maintains chromosome no
...


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Mitosis




Part of the cell cycle
Identical daughter cells produced
Used in asexual reproduction of egg division

Genome: cells total DNA (3m long)
Cell Cycle
Interphase  mitotic phase
Cell physically divides
90% of the cell cycle
Cell grows & copies DNA
Includes G1, S & G2



After DNA duplication the chromosomes condense before cell division
Each chromatid forms 2 sister chromatids with identical DNA

Mitosis

Prophase
Chromosomes
become visible
Spindle forms
Contromeres
move away

Prometaphase
Nuclear
envelope breaks
down
Microtubules
stick to
chromosomes

Metaphase

Anaphase

Telophase

Chromosomes
line up on the
equator

Chromosomes
separate

Identical sets of
chromosomes at
poles

Migrate to poles
Cell becomes egg
shaped

Chromosomes
uncoil

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Meiosis


Sexual reproduction involves:
o Meiosis
o Gamete production
o Fertilization
o It produces variation through different new combinations of alleles

The Role of Meiosis






Life cycle
o Stages in the reproductive history of an organism
o Meiosis alternates with fertilization
Meiosis reduces the chromosome number from diploid (2n) to haploid (n)
This promotes genetic diversity among gametes
Gametes (n) combine by fertilization to produce a zygote (2n)
2n --> meiosis --> n --> fertilization --> 2n

2 nuclear
enevlopes

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Phenotype: the visible characteristics EG seed wrinkle or coat colour
Genotype: the genetic make-up & alleles present
Homozygous: the same EG SS or ss alleles
Heterozygous: different EG Ss alleles

Homologous Pair

Sister Chromatids

Meiosis I


The homologous chromosomes line up together, homologue to homologue, these
homologues then separate

Prophase I


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90% of meiotic division
Homologues pair together
Crossing over occurs at chiasmata
The segments swap and exchange genetic information

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Metaphase I




Chromosomes on the metaphase plate are still in homologous pairs
Kinetochore microtubules attach to one chromosome from each pair

Anaphase I




Homologous pairs are separated
The spindle pulls each chromosome to
opposite poles of the cell
The sister chromatids remain attached

Telophase I




Cytokinesis occurs and two daughter cells are produced
There is no further replication of DNA before the 2nd meiotic division

Meiosis II
Metaphase II
 The chromosomes on the metaphase plate line up on the equator
 The spindle fibres attach to one chromatid from each of the
sisters

Anaphase II


The centromeres finally separate and the sister chromatids
move to opposite sides of the cell

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Telophase II

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

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Separate nuclei form
Cytokinesis occurs
This results in four haploid daughter cells

Transmission Genetics

Segregation


The two alleles for each trait separate during gamete formation (meiosis), then unite at
random, one forms each parent at fertilisation

The Test Cross


Used to distinguish genotype
o Unknown individual is bred with a homozygous recessive

Y y
Y YY Yy
y Yy yy

Dihybrid Cross




How will the alleles be assorted when the double heterozygous F1 plants produce gametes?
o SY & sy only
o SY, Sy, sY & sy?
Mendel saw 4 phenotypes in F2
o Recombinant types appeared
o 9:3:3:1 ratio

Independent Assortment





During gamete formation, different pairs of alleles segregate independently or each other
Not as universal as the law of segregation
o Some genes are linked (on the same chromosome)
o But chromosomes do assort independently during meiosis
Mendel’s law indicated that pairs of genes on homozygous chromosomes segregated during
gamete formation
o True for many
o Some didn’t fit and produced ratios of 3:1 & 9:3:3:1

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Dominance

Incomplete Dominance (i
...
partial or semi dominance)


The heterozygous phenotype is somewhere in between the two homozygous phenotypes
o EG Belgian blue cattle – they have an extreme muscularity gene

Codominance




The phenotype is a mixture of both homozygous phenotypes
o Both alleles are expressed
o EG ABO blood type
o Shorthorn coat colour
 Red or white homozygotes
 Heterozygotes are roan colour
Many alleles are codominant at the molecular level

Pleiotropy


There are multiple effects of a single gene
o EG albino tiger has a white coat and is cross eyed due to one gene

Lethal Alleles




EG Overo allele in horses
o Homozygous foals die within a few days
o They have a white coat and no nerve cells
EG the Manx allele in cats
o Heterozygotes die in utero
o They lack a tail
o 2:1 ratio

Multiple Alleles


Coat colour in rabbits
o One gene has 4 alleles

Multiple Genes







Most traits are controlled by two or more genes and influenced by the environment
o Called multifunctional or polygenic
Some gene interactions further modify phenotype ratios
o EG Epistasis: one gene at one locus influences another gene at a different locus
o Common in control of fur/petal colour
o EG Labrador
 B/b for melanin in skin
 E/e for melanin in hairs
Gene interactions and new combinations can generate hybrid vigour
o Offspring perform better than both pure bred parents
o The influence of multiple genes can produce continuous (quantitate) variation
Environmental variables influence the transmission of a gene: called the phenotype
o Seen in polygenic & single gene traits
o EG Siamese cat coat colour

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Sex Linked Alleles

Chromosome theory of Inheritance


Chromosome movement during meiosis parallels the behaviour of genes

Chromosome Compliment


Each somatic cell contains a set number of chromosomes
o EG 46 chromosomes in humans
o 22 sets of autosomes
o 1 set of sex chromosomes: XY in men or XX in women

Sex Chromosomes




The SRY gene on the Y chromosome ensure that testes and sexual organs develop
The segregation of sex chromosomes into gametes ensures equal numbers of males and
females are produced
The single X chromosome in males must come from the mother

X Chromosome Genes







Males only carry one copy of the X chromosome gene
The pattern of inheritance of these genes differs from that of autosomal genes
Traits determined by these genes are X or sex linked
o EG white & red eyes in fruit flies
Recessive X linked traits are seen more frequently in males than females since only one of the
recessive gene is needed
o This results in different phenotypic ratios
Reciprocal crosses give different results – breaks Mendel’s rules

Linked Genes


Two genes close together on the same chromosome may be independent of each other
o Their alleles aren’t sorted independently
o Dihybrid F2 ratios are atypical
o When chromosomes are far apart on the chromosome, crossing over may break the
line

Sex Limiting Traits



Traits that are only expressed by one sex
The genes are carried by both sexes
o e
...
milk yield
o egg production
o cryptorchidism – unable to grow
o cock feathering

Sex-Inherited Traits


Expressed in both sexes, one sex more often
o EG Sheep horns
o Hip dysplasia in dogs

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Changes in Chromosome Number
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



Disrupts the development
o influence of changes is more substantial than alterations in individual genes
Aneuploidy
o One or more extra / missing chromosomes
o Caused by nondisjunction
o e
...
Trisomy 21 where there is an extra 21 chromosome causing down syndrome
Polyploidy
o Three or more complete sets of chromosomes
o Normal condition in many plants
o Lethal condition in humans and many animals

Mutations in Chromosomes
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Deletions
Duplications
Inversions
Translocations

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Introduction to Molecular Genetics

Proteins





Are central to the phenotype of the organism
They act as:
o Enzymes
o Structural proteins
o Receptors
o Messengers
 Hormones
 Cytokines
Most genes encode a single protein

DNA Form & Function




Genes consist of deoxyribonucleic acid
DNA is two strands of nucleotides
DNA nucleotide:
o Phosphate
o Sugar- deoxyribose
o Base
 Adenine
 Guanine
 Cytosine
 Thymine
o Four different nucleotides in DNA (G, A, T & C)
o They differ only in their base
o The carbons in the sugar are named 1’, 2’, 3’, 4’ and 5’
o The base is linked to the 1’ carbon
o The phosphate is linked to the 5’ carbon

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The nucleotides in DNA are linked by phosphate bonds
(phosphodiester) between the sugars
The 3’ carbon of one sugar is linked to the 5’ carbon of the next
nucleotide
Ends of the DNA strand are called the 5’ and 3’ ends
5’ end: the 5’ carbon of the terminal nucleotide is free
3’ end: the 3’ carbon of the terminal nucleotide is free

A to T: 2 hydrogen bonds
G to C: 3 hydrogen bonds


Structure is called antiparallel because each helical strand travels in
opposite directions

DNA Structure





Right handed double helix: two polynucleotide chains are coiled in a spiral
Sequence of nucleotides are linked by phosphodiester bonds, joining adjacent deoxyribose
groups
The two strands are held together by hydrogen bonding between bases on opposing strands
Purine Is always paired to Pyrimidine

Functions of Genetic Material




Genotypic function: information storage and replication
o Genetic material must store genetic information and accurately transmit that
information from parents or offspring, generation after generation
Phenotypic function: gene expression
o Genetic material must undergo changes so that organisms can adapt to modifications
in the environment
o Without these changed, evolution wouldn’t occur

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Diversity of Life




DNA in all species shows:
o Molecular consistency (similar structure)
o Variation (unique base sequences)
EG:
o Bacteria: 106 nucleotides; one circular
chromosome
o Yeast/fungi:107-108;
linear
chromosomes
o Plants: 108-1011; linear chromosomes
o Mammals: (humans): 3
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11
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The strand of DNA has to be unwound
2
...
Each old strand acts as a template that determines the order of nucleotides to
form the new strand
4
...
i
...
each daughter stand contains
one new and one old backbone
Replication Bubble
These bubbles are produced
where the DNA helix has
unwound to allow for synthesis
of proteins
...

o Each small stretch is called an Okazaki fragment
o DNA polymerase III then synthesises the addition of nucleotides until it reaches the
previous primer
o DNA polymerase I then removes the old
primer and replaces it with DNA
nucleotides
o Between each Okazaki strand there is a
small space, the enzyme DNA ligase
catalyses the formation of the
phosphodiester bond

RNA




This is a nucleotide polymer
Nucleotides of adenine, guanine, cytosine and uracil
Has a sugar of ribose

DNA Mutations






Mutations can occur in DNA
o EG during replication process
o Or due to environmental damage, e
...
radioactive substances
DNA proofreading can correct errors as soon as DNA polymerase makes them
Mismatch repair: scans DNA immediately after it has been replicated and corrects any base
pairing mismatches
Excision repair: removes abnormal bases that have formed because of chemical damage and
replaces them with functional bases

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From Genes to Proteins

Proteins: Functional Gene Products




Genes direct protein production, they dictate every reaction in cells and produce measurable
characteristics
o EG enzymes control chemical reactions
o Genetic polymorphism for the enzyme can lead to differences in enzyme activity EG
Rubisco
Understanding these pathways to protein production enable:
o Precision crop breeding
o Rapid diagnosis of plant or animal health problems

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Location of Gene Protein




Karyon: the cell nucleus
Eukaryotes have a nucleus/nuclear envelope and membrane bound organelles
Prokaryotes don’t have a nucleus and have no membrane bound organelles

Genes

Phenotype

DNA


Transcription

RNA

Translation

Protein

Proteins are synthesised from genes via two processes:
1
...
Translation (protein synthesis)
 Using RNA as a template to synthesise protein

Structure of DNA & RNA





DNA is double stranded, it’s very stable and so doesn’t degrade
RNA is single stranded
There is no thymine in RNA, uracil replaces it
RNA nucleotides have an additional O atom

Transcription




3 essential components
o Protein coding gene
 Important structural features that inform the building of RNA
 Promoter region: specific DNA sequence where transcription begins
 Coding sequence: specific DNA sequence, coding information for the protein
 Terminator: a specific DNA sequence that determines the end of the RNA
transcript (in prokaryotes)
o RNA polymerase
 An enzyme to catalyse the building of RNA from DNA
 Also acts as a primase that initiates DNA replication (aka transcriptase)
 It has 5 different polypeptides, two alpha, two beta and a sigma copy, this is
called the Halo enzyme, it initially binds onto the DNA promoter region, the
DNA helix is then unwound and transcription can begin
o NTPs (nucleoside Triphosphates)
 The building blocks of the new RNA
 A nucleoside is the base attached to the ribose
 Uridine, cytidine, adenosine, guanosine are the 4 different nucleosides
3 stages:
o Initiation
 Occurs at the promoter region
 RNA polymerase binds to the promoter and begins to unwind the DNA
 As the DNA and RNA transcript exits the RNA polymerase, the RNA is removed
from the DNA template and the DNA rewinds together
o Elongation
o Termination

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



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When the RNA polymerase reaches the termination site, the RNA transcript
is released from the template
 RNA polymerase dissociates from the DNA and can perform other rounds of
transcription
mRNA carries the gene sequence for translation of the protein production
Other RNAs also produced by transcription, involved in protein production
The type of RNS produced depends on the DNA sequence used to transcribe it
o Different DNA sequences encode different types of RNA

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RNA Processing

Eukaryotic Promoters


There are 3 phases of transcription:
o Initiation: occurs at the promoter region, the RNA polymerase binds to the promoter
and begins to unwind the DNA
 TATA box: a DNA sequence within the promoter region of DNA, rich
in AT base pairs



o
o

Transcription factors: proteins the bind to the DNA and influence
transcription
 One transcription factor recognises the TATA box and binds and the
additional TFs recognise the bound TF and bind
 RNA polymerase recognises the transcription factors which enable it
to bind to the DNA in the correct place and direction
 RNA polymerase: with a sigma subunit
 The additional transcription factors bind to the DNA using RNA
polymerase, the transcription initiation complex
 The RNA polymerase then unwinds the double helix and RNA
synthesis begins at the start codon on the template strand
Elongation
Termination
 When RNA polymerase reaches the termination site, the RNA transcript is
released from the template
 Polyadenylation signal sequence: this is a DNA sequence within the
termination site region of the DNA, this codes for the polyadenylation signal
in the mRNA, usually AAUAAA
 Enzyme: this recognises the polyadenylation signal and releases the mRNA
from RNA polymerase

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RNA Processing
Characteristic
Site of transcription & Translation
Gene structure
Modification of mRNA after
transcription before translation

Prokaryotes
Cytoplasm
Complementary to
protein structure
None

Eukaryotes
Transcription – nucleus; Translation
- cytoplasm
Noncoding sequences “introns”
A) Additions to mRNA ends
B) Intron removal



A) additions to mRNA ends
I
...
3’ tail
 this is added immediately after the mRNA transcript has been released from
RNA polymerase
 Called a ‘Poly A Tail’ which is 100-300 adenine nucleotides long
 Enables the mRNA to be exported from the nucleus to the cytoplasm
 Helps to promote mRNA stability and prevent degradation



B) intron removal (splicing)
 Eukaryotic gene sequences contain non-coding sections that don’t code for a protein
 These are transcribed into mRNA sequences which produces introns
 Small nuclear Ribonucleoproteins (snRNPs): these are proteins that perform the
splicing
 RNA polymerase transcribes both the introns and exons, this material is called the
pre-mRNA
 For the mRNA to leave the nucleus for translation to occur in the cytoplasm the
introns need to be removed

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

Splicing means to form connections



Things needed for RNA splicing:
 Pre-mRNA
 snRNPs
o At the boundaries between introns & exons there are consensus
sequences which are short stretches of DNA that appear in many
different genes, this is complementary to the consensus sequence on
the 5’ boundary and so it binds by base pairing

o

o
o
o

Other snRNPs also attach at
additional point along the premRNA
These then join together to form
a spliceosome which will loop out the intron
The Spliceosome cuts the pre-mRNA at the intron-exon boundary
RNA Splicing
I
...

The free 3’OH group at the end of the cut exon reacts with
the 5’ phosphate of the other exon
III
...

The excised intron is degraded in the nucleus & recycled

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Translation

Translation

Codons




These are a series of three nucleotides in mRNA
that codes for an amino acid
Codons direct the placement of specific amino
acids into a protein
There are 64 codons, 3 functions as stop signals
marking where ribosomes end translation, there are
20 amino acids and 1 start codon

Translation Stages


Initiation
1
...
Prokaryotes: a recognition
sequence (AGGAGG)
b
...
Methione
charge
tRNA-containing
complementary sequence to start codon
(anticodon, UAC) binds to the start codon (AUG)
 The first amino acid in the polypeptide
chain is methionine
 The combination of mRNA, ribosome
small subunit and tRNA forms the
initiation complex
3
...
A: anticodon
b
...
E: exit
Methionine charged tRNA occupies the site
The A site is aligned with the next mRNA codon (CCG)

Elongation
1
...
Peptide bond formation:
 Proline is linked to methionine by
peptidyl transferase activity of the
ribosome large subunit
 Peptidyl transferase has catalytic
activity for 2 reactions:
a
...
Forms a bond between that
amino acid and the new amino
acid attached to its tRNA in the
A site
3
...
The process then repeats
 The free site A attracts complementary charged anticodon (AUA) for the
codon in the A site
 The bond between the amino acids and tRNA in the P site breaks
 These amino acids bond to the new amino acid
on the tRNA in the A site
 The ribosome then moves along the mRNA to
the next codon, the old tRNA is release and the
new tRNA is in the P site
Termination
1
...
The release factor disconnects the polypeptide from the
tRNA in the P site
3
...
Stay within the cell: complete translation and be released to an organelle, or remain
in the cytosol
 Proteins contain signal sequence that direct them to the nucleus,
mitochondria, plastids or peroxisomes, if there is a lack of signal sequence
they will remain in the cytosol
2
...
Most proteins are not identical to the polypeptide chains
2
...
This is essential to the final function of the protein
 Proteolysis: cutting the polypeptide allowing the fragments to fold into
different shapes
 EG cutting the signal sequence from the growing polypeptide chain in
the ER
 Some proteins made from polypeptides are cut into their final
products by proteases
 Proteases are essential to some viruses (EG HIV) and larger viral
polypeptides can’t fold without being cut
 Some drugs for AIDS work by inhibiting HIV protease, which prevents
the formation of proteins needed for viral reproduction



Glycosylation: the addition of sugars to the polypeptide, important for
targeting and recognition
 The addition of sugars forms glycoproteins
 It is catalysed by enzymes in the ER and Golgi apparatus
 This is essential for directing proteins to lysosomes and important in
the conformation of proteins and their recognition at the cell surface



Phosphorylation: the addition of phosphate groups to alter the shape of the
protein

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Catalysed by protein kinases
Charged phosphate groups change the conformation of a protein
which often exposes the active site of an enzyme or binding site to
another protein
Important for cell signalling

Transport to Cellular Destination
1
...
Or the translation is stopped and it is moved to the ER to finish synthesis

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Regulation of Gene Expression

Regulation of Gene Expression




Temporal variation in gene expression and protein synthesis EG butterflies
Organisms go through development stages produced by different sets of proteins, controlled
by regulated gene expression
Regulating gene expression also allows organisms to respond to changes in their environment
o EG plant in a drought
 Plants must simultaneously produce multiple proteins whose genes are
scattered throughout the genome (different chromosomes)
 The synthesis of these proteins is called ‘stress response’
 To coordinate expression, each stress gene has a specific regulatory sequence
near its promoter called the stress response element (SRE)
1
...
The TF binds to the SRE of each gene, which stimulates the
transcription of each gene that has the same SRE sequence,
3
...
E
...
Glucose =, the preferred energy source is the easiest
sugar to metabolise or lactose that is composed of βglucose which is more complex to metabolise
3
...
Functional genes: genes for enzymes involved in
lactose metabolism
 Activator Protein: positive regulation

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o

o
o
o
o

The binding of the activator protein stimulates transcription
to allow for the RNA polymerase to bind to the promoter
region
o When glucose levels are low, positive regulation can increase
the efficiency of lac operon transcription (not turned on
when glucose levels are high)
 Two environments affect the rate of lac operon:
o Low glucose (when lactose is also present)
1
...
RNA polymerase then binds more efficiently to the
promoter
3
...
cAMP is low, CRP is inactive and does not bind to the
promoter, RNA polymerase is unable to bind
efficiently
2
...
Bind to the enhancer and regulatory protein sites
 2
...
11
...
They cleave double
stranded DNA at a position either within or outside the recognition site
This recognition site can be of 4, 6 or 8 nucleotides long
Restriction enzymes can release:
- Cohesive/sticky ends
- Blunt ends
 There are more than 3600 restriction enzymes

o

Modifying enzymes
 Methyltransferases
 This catalyses the transfer of a methyl (CH3) group to DNA bases (N6Methyladenine, N4-Methylcytosine, C5-Methylcytosine)
 Used to block restriction sites
 Approximately 1% of DNA bases undergo DNA methylation
 Nucleases (DNases, RNases)
 Enzymes that cleave randomly nucleic acids
 Deoxyribonucleases, DNases: nucleases that cleave single stranded or
double stranded DNA
o Endonucleases: cut inside the sequence
o Exonucleases: cut from the extremities
 Ribonucleases, RNases: nucleases that cut the RNA
 DNA ligases
 These catalyse the formation of a phosphodiester bond between the
5’ phosphate of one DNA fragment & the 3’ hydroxyl of another

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



There are two types:
o ATP-dependent DNA ligases
o NAD-dependent DNA ligases
 T4 DNA ligase is used in cloning to ligate DNA fragments
Polymerases










These copy a DNA strand into another DNA strand
There are 2 domains and 3 functions:
o C-ter
...
Small fragment has: 5’ to 3’ exonuclease activity
There are 3 main types of DNA polymerases in bacteria:
o DNA pol I: main enzyme for DNA replication in bacteria, the
DNA polymerases use in PCR belong to this group
o DNA pol II: involved in DNA repair
o DNA pol III: involved in DNA replication
Processivity: number of nucleotides added to the new strand per
second
Fidelity: the rate of errors (wrong nucleotides added)
RNA Polymerases

o
o



These transcribe single stranded DNA into RNA
In prokaryotes: the same RNA polymerase produces
messenger RNA and non-coding RNA (rRNA, tRNA, sRNA)
o In eukaryotes:
 RNA polymerase I: large ribosomal RNAs
 RNA polymerase II: messenger RNA
 RNA polymerase III: transfer RNA and small RNA
 Mitochondrial & chloroplast RNA polymerases
Reverse transcriptase



RBA dependent DNA polymerase

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Transcribe single-stranded RNA into single-stranded complementary
DNA (cDNA)
Used mainly by retroviruses (EG HIV, AMV)
In the lab Reverse Transcriptase are used to produce in vitro cDNA
from RNA

Molecular tools
o Vectors
 They are small DNA molecules having regulatory and coding sequences
 Foreign DNA can be inserted into them
 They are used as carriers of foreign DNA host cells
 Origin of replication: replication of the vector, together with the foreign DNA
fragment inserted into it
 Genetic markers: selection of cells which have taken up the plasmid DNA
 Multiple cloning site: a site where DNA is inserted
 Transfer DNA (some vectors): transfer a gene into a target genome, vector
DNA can be used as a DNA vaccine
o Plasmids
 Double stranded circular bacterial DNA used for molecular cloning to amplify
or express insert DNA into bacterial hosts
 Phagemids or phasmids
 DNA cloning vectors derived from phage DNA and containing an
origin of replication
...
They are used to carry large insert DNA up to 300kb
and are used to create and store genomic libraries
...
11
...
6 and 2
...
The charged molecules are first retained on a resin having the opposite charge
to them
2
...
The retained molecules are then eluted using a salt solution
Size-Exclusion chromatography
o Separates molecules based on their size
o The small molecules are retained in the resin particles
o Large molecules pass through between the resin particles

DNA & RNA Detection






Fluorescent intercalating agents:
o Ethidium bromide: orange fluorescence under UV light
o SYBRGreen: green fluorescence under UV light
Dyes: Hoechst & silver
Radioactive labelling: 32P(ATP)
Fluorescent labelling: Cy3, Cy5, 6-FAM, Rox, TAMRA, etc
...
Each spot contains a probe for a gene
The chips are hybridised with DNA or RNA samples for detection and quantification of
particular sequences
Macro chips:
o Contain hundreds of macroscopic spots (Probes) on glass or membrane supports
Microchips:
o Contain thousands of microscopic spots (probes) on glass
supports

DNA Sequencing








Sanger & Coulson method (termination method, 1977):
o This method used dideoxynucleotides (ddATP, ddTTP,
ddGTP and ddCTP) for early termination of DNA
polymerisation
Sequencing reaction:
1
...
A primer is required
3
...
Labelled ddNTPs (ddATP, ddTTP, ddGTP
and ddCTP) (big dye terminator) are
required
5
...
11
...
The double stranded DNA is denatured
in 95°C
2
...
The primers are extended by the
attachment of free nucleotides
4
...
Initial denaturation
2
...
Annealing temperature and time are depended
of the type of primer
4
...
12
...
The DNA is digested with the appropriate
restriction enzymes
2
...
Different DNA fragments can be successively
ligated to produce rDNA
4
...
Complementary DNA (cDNA) libraries
 Collections of bacterial clones containing cDNA representing expressed genes
in a given tissue at specific conditions and corresponding to a specific
physiological situation
 Only expressed genes are represented
2
...
RNA extraction and mRNA purification
2
...
Ligation of the adaptors and their digestion
4
...
Packing of he rDNA (cDNA in the vector) into the phage capsule
6
...
gDNA extraction & purification
2
...
Ligation of DNA fragments into the digested vector
4
...
Plating the phage library into host bacteria
Screening libraries

Probes


Labelled RNA or DNA fragments with a known sequence used to detect a complementary
sequence in a DNA or RNA population




Homologous probes: from the same organism
Heterologous probes: from a different organism, used to detect similar sequences in the
studied organism
The isolated clone is then sequenced and the sequence is analysed



Genomics: Genome Analysis



Sequencing of the transcriptome (total expressed genes in a tissue) or the entire genome
Transcriptome analysis:

Introduction to Genetics

ACE1013

o



Expressed sequence tags (ESTs): random sequencing of thousands of clones from a
cDNA library prepared from a given tissue at specific conditions
Genome analysis:
o Sequencing DNA libraries containing fragments representing the entire genome of a
given organism
o The obtained sequences are made available to the scientific community via databases

Genome Sequences







Coding sequences
Regulatory sequences
Mapping genomes
o Assigning/locating a specific gene to a particular region of a chromosome and
determine the location and relative distances between genes on the chromosome
Physical map:
o The physical, DNA-base-pair distances from one gene to another
Genetic linkage map:
o Order of genes on a chromosome and the relative distances between these genes
o Tracing the inheritance of multiple gene traits

Associating Gene with Function/Phenotype




Associating change in gene expression with function
o Single gene approach
 Northern blotting
 Quantitative reverse transcription-polymerase chain reaction (QRT-OCR)
o Global approach
 Checking the expression of thousands of genes at the same time
 Microarray technology
 RNA sequencing
Gene silencing with function
o Knock down technologies
 Antisense RNA
 Used to reduce the expression of a gene to check the impact on the
physiology
 Antisense RNA are RNA sequences complementary to the target RNA,
they hybridise to it and block its translation
 They are produced by transforming the target organism with a cDNA
that is transcribed into antisense RNA
 RNA interference using small RNA
 Small RNA are natural or synthetic double stranded RNA molecules of
20-15 nucleotides complementary to the target RNA
 They hybridise to the target RNA and block its translation, they lead
to its degradation by specific RNases
 Antisense RNA and siRNA allow to identify gene function

Introduction to Genetics

o

Knock-out Technology
 Knock-out are collections of insertional mutants produced by rDNA
technology
 A vector is used to transfer randomly or specifically a tDNA to a location in
the genome to alter a gene at that locus
 Each mutant has an alteration in a single gene
 By tracing the tDNA it is possible to find which gene was affected
 By identifying the change in the phenotype it is possible to link gene with
function

4
...
15
Transgenesis


ACE1013

The transfer of a gene from
one species to another species
o Direct transfer
o Semi-direct transfer
o Mediated transfer

Recombinant DNA Technology II

Introduction to Genetics




Methods used for DNA transfer
o Transformation/Transfection:
 This method uses competent cells induced chemically (Lithium Acetate) to
take in the DNA
 The DNA is added to competent cells and left to
enter the cells on ice
 Usually a heat shock (42°C) is required to complete
the DNA transfer
o Electroporation
 This method uses an electric pulse to drive DNA into
cells
 DNA is added to cells and placed in a cuvette having electrodes on two sides
 An electric pulse is applied to the electrodes using a pulse power supply
o Microinjection
 This method involves the injection of naked DNA into cells
using a pipette that has a microscopic tip
 It is mainly used for transforming animal cells (eggs)
 Done under a microscope
o Bombardment
 This method uses a gene gun to transfer DNA coated on gold particles into
host tissues
 The gene gun uses compressed helium, which is pulse to project the gold
particles into the cells
Methods using Mediated DNA Transfer
o Liposome mediated transfer
 The DNA is packaged into liposomes which fuse to the
plasma membrane releasing the DNA inside the cell
o Bacterial and Viral Mediated Transfer
 DNA is introduced in bacterial or viral strains which
infect the target cells and transfer DNA into their genome
 Agrobacterium tumefaciens: is a bacterial strain capable of transferring DNA
to many plant species
 Vaccine virus: transfers DNA to different animal and human cell types
o Selective breeding
 This method is used to transfer DNA from one species to a close species via
successive crosses using a number of intermediates

Applications of rDNA Technology


ACE1013

Production of recombinant proteins:
o The production of proteins using an expression
system transformed with rDNA containing the
coding sequence for the protein of interest
o Expression systems:
 Host cells together with compatible
expression vectors allowing high
expression of recombinant proteins in
these cells

Introduction to Genetics

ACE1013

o







Hosts:
 Bacteria: Escherichia coli
Rapid growth
Inducible system
 Yeast: Saccharomyces ceriviseae
Eukaryotic protein folding system
Good for producing functional recombinant eukaryotic proteins
Protein Production systems

o The recombinant protein can represent up to 50% of proteins produced by the cell
Protein secretion: no extraction:
o A single peptide (SP) is usually required for protein secretion outside the cells
o The nucleotide sequence encoding the SP is added to the sequence encoding the
recombinant protein in the rDNA
Example of therapeutic Recombinant Proteins
o Insulin: hormone controlling glucose uptake by cells EG diabetes
o Growth factor: used by young people with slow growth
o Coagulation factors: used by haemophiliac patients
o Recombinant vaccines

Molecular Pharming




The production of pharmaceuticals in plants and
animals
o High production systems
o Low production costs
o Possibility for integrating pharmaceuticals
in food
o Lower risk
Pharming in plants:
o Many plant systems are easy to transform
o Highly productive systems
o Low maintenance and low cost
o No possibility of disease transfer to humans
o It is possible to produce pharmaceuticals in seeds: the possibility of storage at room
temperature for many years
 Proteins would remain stable for many years

Introduction to Genetics


ACE1013

 Easy to store
Pharming in Animals:

Genetically Modified Organisms








Organisms who’s genome was
modified via the insertion of rDNA to
improve their phenotype
GM Plants:
o GM crops represent about 30% (increasing) of the world’s total crop production
...
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