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Corso di Struttura e Funzione dei Genomi (LM7) mutuato con il corso di Genomica e Trascrittomica (LM9)
Genomi
Diapositive, articoli e dispense:
http://profs.sci.univr.it/delledonne/Insegnamenti/Index.html
Vettori di clonaggio
I vettori di clonaggio sono brevemente trattati ai soli fini di un «ripasso» delle loro caratteristiche,
dato che ad essi si fa riferimento in diversi argormenti trattati nell’ambito del Corso
Per lo studente con scarse conoscenze, informazioni dettagliate sono disponibili su:
http://profs.sci.univr.it/~delledon/Insegnamenti/Dispense Tecnologie Biomolecolari 2010.pdf
Plasmidi
Cosmidi
Fagi
Fagi
Yeast Artificial Chromosome (YAC)
BAC
Il vettore BAC è costituito da:
 geni oriS e repE (Mediano le replicazione unidirezionale
del fattore F)
 geni parA e parB (Mantengono il numero di copie del
fattore ad un basso livello (1 copia) .Un alto numero di
copie potrebbe causare problemi di instabilità degli
inserti per delezioni e riarrangiamenti del DNA clonato
a causa di eventi di ricombinazione omologa)
 gene per la resistenza ad un antibiotico
 sito di clonaggio: È fiancheggiato dai promotori T7 e
SP6 che generano sonde di DNA per il chromosome
walking e per il sequenziamento d'inserti di DNA. cosN
e loxP sono siti di taglio specifici per terminasi del
batteriofago l e consentono la formazione di estremità
che possono essere usate, nelle mappe di restrizione, per
riarrangiare i cloni. I cloni ricombinanti sono selezionati
mediante colorazione bianco/blu.
Comparazione fra YAC and BAC Cloning System
Features
YAC
BAC
Configuration
Linear
Circular
Host
Yeast
Bacteria
Copy Number / Cell
1
1-2
Cloning Capacity
Unlimited
up-to 350 kb
Transformation
Spheroplast (10^7
T/ug)
Electroporation (10^10
T/ug)
Chimerism
up to 40%
None to low
DNA Isolation
Pulsed-field-gelelectrophoresis Gel Isolation
Standard Plasmid
Miniprep
Insert Stability
Unstable
Stable
Mappe genetiche e fisiche
Le mappe sono brevemente trattate ai soli fini di un «ripasso» delle loro
caratteristiche, dato che ad esse si fa riferimento in diversi argormenti
trattati nell’ambito del Corso
Mappe fisiche e mappe genetiche
Mappa genetica o Mappa di concatenazione: Rappresentazione della
distanza che separa i geni, basata sui dati di ricombinazione genetica
Mappa fisica: Rappresentazione della distanza che si basa sulla distanza
fisica lungo il cromosoma (sovente espressa in bp)
Relazione
tra
mappa
genetica
e
mappa
fisica
La distanza della mappa genetica per le 3000Mb del genoma umano e' di
circa 3000cM e pertanto 1cM corrisponde approssimativamente a una
distanza di mappa fisica di 1Mb.
In realta' il rapporto delle distanze di mappa genetica e fisica sui segmenti
cromosomici spesso deviano da questo valore medio a causa della
localizzazione non casuale dei chiasmi. I segmenti cromosomici contenenti gli
"hot spots" di ricombinazione mostrano una piu' alta frequenza di
ricombinazione e per tanto marcatori molecolari localizzati in questa zona
sembrano piu' distanti del reale. In generale c'e' una frequenza di crossingover piu' alta in corrispondenza delle regioni subtelomeriche rispetto a quelle
centromeriche.
Mappa genetica
Mappa fisica
Overview genetic markers
Molecular markers
abundance
level of
polymorphism
locus specificity
codominance
of alleles
reproducibility
labor-intensity
Allozymes
low
low
yes
yes
high
low
RFLP
high
medium
yes
yes
high
high
Minisatellites
medium
high
no/yes
no/yes
high
high
PCR-sequencing
low
low
yes
yes
high
high
RAPD
high
medium
no
no
low
low
Microsatellites
high
high
yes
yes
high
low
ISSR
medium-high
medium
no
no
medium-high
low
SSCP
low
low
yes
yes
medium
low-medium
CAPS
low
low-medium
yes
yes
high
low-medium
SCAR
low
medium
yes
yes/no
high
low
AFLP
high
medium
no
no/yes
high
medium
TaqMan
low
yes
no/yes
high
low
RAD
Isoenzymes (http://www.cgn.wageningen-ur.nl/pgr/)
Allozymes are allelic variants of enzymes encoded by structural genes. Enzymes are proteins consisting of amino acids,
some of which are electrically charged. As a result, enzymes have a net electric charge, based on the stretch of amino acids
comprising the protein. When due to mutation an amino acid has been replaced, the net electric charge of the protein may
have been altered. Because changes in electric charge affect the migration rate of proteins in an electric field, allelic variation
can be detected by gel-electrophoresis and subsequent specific enzymatic staining. Per enzyme usually two or more loci can
be distinguished that have been termed isoloci. Therefore, allozyme variation is also referred to as isozyme variation.
Synonyms
•Isozymes
Analytical procedures
•Preparation of tissue homogenates
•Separation of the polymorphisms by polyacrylamide or starch gel-electrophoresis
•Visualization of the polymorphisms by specific enzymatic staining
Main Requirements
•Laboratory setup for gel-electrophoresis
•Facilities/equipment to maintain gels at 2-5 °C during electrophoresis
Advantages
•No DNA extraction involved
•No primers or probes required
•Quick and easy to assay
•Low costs involved
•Zymograms can be interpreted in terms of loci and alleles
•Codominance of alleles
•High reproducibility
Disadvantages
•Low abundance
•Low level of polymorphism
•Zymograms sometimes difficult to interpret due to complex banding profiles
•Proteins with identical electrophoretic mobility (co-migration) may not be homologous
•Profilo enzimatico influenzato dall’ambiente (es. perossidasi indotte in condizioni di stress ossidativo)
RFLP (Restriction Fragment Length Polymorphism)
RFLPs are fragments of restricted DNA (usually within the 2-10 kb range) separated by gel electrophoresis and detected by
subsequent Southern blot hybridization to a radiolabeled DNA probe consisting of a sequence homologous to a specific chromosomal
region. The locus specific probes, consisting of a sequence of unknown identity or part of the sequence of a cloned gene, are
obtained by molecular cloning and isolation of suitable DNA fragments. Sequence variation affecting the occurrence (absence or
presence) of endonuclease recognition sites is considered to be main cause of length polymorphisms. RFLPs are generally found to
be moderately polymorphic and can be applied in comparisons ranging from the individual level to closely related species. Because
of their high genomic abundance and random distribution throughout the genome, RFLPs have frequently been used in gene
mapping studies.
Analytical procedures
•Extraction of DNA
•Digestion of DNA by endonuclease restriction
•Separation of the fragments by agarose gel-electrophoresis
•Transference of the fragments to a nylon filter by Southern blotting
•Hybridization of fragments to a locus specific radiolabeled DNA probe
•Visualization of the polymorphisms by autoradiography
Advantages
•High genomic abundance
•Random distribution throughout the genome
•Band profiles can be interpreted in terms of loci and alleles
•Codominance of alleles
•High reproducibility
•Filters can be washed and reprobed several times
Disadvantages
•Large quantities of purified, high molecular weight DNA required (5-10 µg)
•Laborious and technically demanding
•High costs involved
•Additional development costs in case suitable probes are unavailable
•Not amenable to automation
RFLP (restriction fragment length polymorphism )
RFLP (restriction fragment length polymorphism )
CAPS (Cleaved Amplified Polymorphic Sequence)
CAPS are DNA fragments amplified by the Polymerase Chain Reaction (PCR) using specific 20-25 bp primers, followed by
digestion with a restriction endonuclease. Subsequently, length polymorphisms resulting from variation in the occurrence of
restriction sites are identified by gel-electrophoresis of the digested products. In comparison with RFLP-analysis, polymorphisms
are more difficult to identify because of the limited size of the amplified fragments (300-1800 bp). CAPS-analysis, however, does
not require time-consuming Southern blot hybridization and radioactive detection. CAPS have been applied predominantly in gene
mapping studies.
Analytical procedures
•Extraction of DNA
•Amplification of DNA fragments by PCR
•Digestion of the amplified fragments by endonuclease restriction
•Separation of the fragments by agarose or polyacrylamide gel-electrophoresis
•Visualization of the polymorphisms by ethidium-bromide staining and ultraviolet light (agarose gels) or silver staining
(polyacrylamide gels)
Advantages
•Low quantities of template DNA required (50-100 ng per reaction)
•Band profiles can be interpreted in terms of loci and alleles
•Codominance of alleles
•High reproducibility
Disadvantages
•Sequence data required for primer construction
marker B4 is a cleaved amplified polymorphism sequence (CAPS) marker.
RAPD (Random Amplified Polymorphic DNA)
RAPDs are DNA fragments amplified by the Polymerase Chain Reaction (PCR) using short (generally 10 bp) synthetic primers of
random sequence. These oligonucleotides serve as both forward and reverse primer and usually are able to amplify fragments from
3-10 genomic sites simultaneously. Amplified fragments (within the 0.5-5 kb range) are separated by gel-electrophoresis and
polymorphisms are detected as the presence or absence of bands of particular size. These polymorphisms are considered to be
primarily due to variation in the primer annealing sites. RAPDs have been used for many purposes, ranging from studies at the
individual level (e.g. genetic identity) to studies involving closely related species. Due to their very high genomic abundance, RAPDs
have also been applied in gene mapping studies.
Analytical procedures
•Extraction of DNA
•Amplification of DNA fragments by PCR
•Separation of the polymorphisms by agarose gel-electrophoresis (AP-PCR and DAF fragments are usually separated on
polyacrylamide gels)
•Visualization of the polymorphisms by ethidium-bromide staining and ultraviolet light (AP-PCR and DAF fragments are usually
visualized by silver staining or autoradiography)
Advantages
•Low quantities of template DNA required (5-50 ng per reaction)
•No sequence data for primer construction required
•Low costs involved
•Very high genomic abundance
•Random distribution throughout the genome
•Generation of multiple bands per reaction
•Amenable to automation
Disadvantages
•Purified, high molecular weight DNA required
•Precautions are needed to avoid contamination of DNA because short, random primers are used that are able to amplify DNA
fragments in a variety of organisms
•Highly standardized experimental procedures are needed because of sensitivity to reaction conditions
•Dominance of alleles
•Low reproducibility
•Similar sized fragments may not be homologous
RAPD (Random amplified polymorphic DNA )
RAPD (Random amplified polymorphic DNA )
SCAR (Sequence Characterized Amplified Region)
SCARs are DNA fragments amplified by the Polymerase Chain Reaction (PCR) using specific 15-30 bp primers, designed from
nucleotide sequences established in cloned RAPD (Random Amplified Polymorphic DNA) fragments linked to a trait of interest. By
using longer PCR primers, SCARs do not face the problem of low reproducibility generally encountered with RAPDs. SCARs are
locus specific and have been widely applied in gene mapping studies and marker assisted selection.
Analytical procedures
•Extraction of DNA
•Amplification of DNA fragments by PCR
•Separation of the polymorphisms by agarose gel-electrophoresis
•Visualization of the polymorphisms by ethidium-bromide staining and ultraviolet light
Advantages
•Low quantities of template DNA required (10-100 ng per reaction)
•Easy and quick to assay
•High reproducibility
•Amenable to automation
Disadvantages
•Sequence data required for primer construction
AFLP (Amplified Fragment Length Polymorphism)
AFLPTM is a trademark of Keygene (Wageningen). AFLPs are DNA fragments (80-500 bp) obtained from endonuclease restriction,
followed by ligation of oligonucleotide adapters to the fragments and selective amplification by the Polymerase Chain Reaction
(PCR). The PCR-primers consist of a core sequence (part of the adapter), a restriction enzyme specific sequence and 1-3 selective
nucleotides. The AFLP-technique simultaneously generates fragments from many genomic sites (usually 50-100 fragments per
reaction) that are separated by gel-electrophoresis and generally scored as a dominant marker. However, by using automatic gel
scanners, heterozygotes may be distinguished from homozygotes based on band intensity differences. Because of the highly
informative fingerprinting profiles generally obtained, AFLPs can be applied in studies involving genetic identity, parentage and
identification of clones and cultivars. Due to their high genomic abundance and random distribution throughout the genome, AFLPs
are also considered relevant markers in gene mapping studies .
Analytical procedures
•Extraction of DNA
•Digestion of DNA by endonuclease restriction and ligation of oligonucleotide adapters to the DNA fragments
•Selective amplification of part of the DNA fragments
•Separation of the fragments by polyacrylamide gel-electrophoresis
•Visualization of the polymorphisms by autoradiography, silver staining or fluorescence
Advantages
•No sequence data for primer construction required
•High genomic abundance
•Random distribution throughout the genome, although clustering around centromers has been reported
•Generation of many informative bands per reaction
•High reproducibility
•Amenable to automation
Disadvantages
•Purified, high molecular weight DNA required
•Dominance of alleles
•Similar sized fragments may not be homologous
Amplified Fragment Length Polymorphisms (AFLPs)
SSCP (Single-Strand Conformation Polymorphism)
SSCPs are DNA fragments (200-800 bp) amplified by the Polymerase Chain Reaction (PCR) using specific 20-25 bp primers,
followed by gel-electrophoresis of single-strand DNA to detect nucleotide sequence variation. The method is based on the fact that
the electrophoretic mobility of single-strand DNA highly depends on the secondary structure (conformation) of the molecule, which
changes significantly in case of mutation. SSCP provides a method to detect nucleotide variation without the need to sequence DNA
samples. SSCPs have been applied to detect mutations in genes using gene sequence information for primer construction .
Related techniques
•DGGE (Denaturing Gradient Gel Electrophoresis): separation of DNA fragments due to mobility differences under increasing
denaturing conditions (formamide/urea concentrations)
•TGGE (Thermal Gradient Gel Electrophoresis): similar to DGGE, but subjected to heath-denaturing conditions
Analytical procedures
•Extraction of DNA
•Amplification of DNA fragments by PCR
•Denaturation of the amplified fragments to a single-strand form
•Separation of the polymorphisms by polyacrylamide gel-electrophoresis
•Visualization of the polymorphisms by silver staining or autoradiography
Advantages
•Low quantities of template DNA required (10-100 ng per reaction)
•Band profiles can be interpreted in terms of loci and alleles
•Codominance of alleles
Disadvantages
•Sequence data required for primer construction
•Highly standardized electrophoretical conditions are needed to obtain reproducible results
•Absence of mutation cannot be proven because some mutations may remain undetected
Denaturing Gradient Gel Electrophoresis
(DGGE)
PCR primer with a 5' tail consisting of a sequence of 40 GC!
Microsatellites
Microsatellites are molecular marker loci consisting of tandem repeat units of very short (1-5 basepairs) nucleotide motif. In
case the nucleotide sequences in the flanking regions of the microsatellite are known, specific primers (generally 20-25 bp) can be
designed to amplify the microsatellite by the Polymerase Chain Reaction (PCR). Polymerase slippage during DNA replication (or
slipped strand mispairing) is considered to be the main cause of variation in the number of repeat units, resulting in length
polymorphisms that can be detected by gel-electrophoresis. Due to their high level of polymorphism, microsatellites are informative
markers that can be used for many population genetic purposes, ranging from the individual level (e.g. clone and strain identification)
to closely related species.
Synonyms
•SSLP (Simple Sequence Length Polymorphisms); SSR (Simple Sequence Repeats); STMS (Sequence Tagged Microsatellites)
Analytical procedures
•Extraction of DNA
•Amplification of DNA fragments by PCR
•Separation of the polymorphisms by polyacrylamide Visualization of the polymorphisms by autoradiography, silver staining or
fluorescence (polyacrylamide gels), or ethidium-bromide staining and ultraviolet light (agarose gels)
Advantages
•Low quantities of template DNA required (10-100 ng per reaction)
•High genomic abundance
•Random distribution throughout the genome
•High level of polymorphism
•Codominance of alleles
•Allele sizes can be determined with an accuracy of 1 bp, allowing accurate comparison across different gels
•High reproducibility
•Different microsatellites may be multiplexed in PCR or on gel
•Wide range of applications
•Amenable to automation
Disadvantages
•High development costs in case primers are not yet available
•Heterozygotes may be misclassified as homozygotes when null-alleles occur due to mutation in the primer annealing sites
Microsatelliti
Microsatellite DNA is thus a class of repetitive DNA based
on dinucleotide repeats. The most common type consists of
repeats of CA and its complement GT, as in the following
example:
Figura SSR
When the repeating unit is less than four, the VNTR is called a microsatellite and when the repeating unit is longer it is a minisatellite.
I microsatelliti sono anche chiamati Simple Sequence Repeat (SSR)
Minisatellites
Minisatellites are molecular marker loci consisting of tandem repeat units of a 10-50 base motif, flanked by conserved
endonuclease restriction sites. They are detected by gel electrophoresis of restricted DNA and subsequent Southern blot
hybridization to a radiolabeled DNA probe containing multiple copies of the minisatellite core sequence. A minisatellite profile
consisting of many bands (within the 4-20 kb range) is generated by using common multilocus probes that are able to hybridize to
minisatellite sequences in different species. Locus specific probes can be developed by molecular cloning of DNA restriction
fragments and subsequent screening with a multilocus minisatellite probe. Variation in the number of repeat units is considered to be
the main cause of length polymorphisms. Due to the high mutation rate of minisatellites, the level of polymorphism is substantial,
generally resulting in unique multilocus profiles. Therefore, minisatellites are particularly useful in studies involving genetic identity,
parentage, clonal growth and structure, and identification of varieties and cultivars
Synonyms
•DNA fingerprinting; VNTR (Variable Number of Tandem Repeats)
Analytical procedures
•Extraction of DNA
•Digestion of DNA by endonuclease restriction
•Separation of the fragments by agarose gel-electrophoresis
•Transference of the fragments to a nylon filter by Southern blotting
•Hybridization of fragments to a radiolabeled minisatellite probe
•Visualization of the polymorphisms by autoradiography
Advantages
•High level of polymorphism
•Generation of many informative bands per reaction (in case of multilocus probes)
•High reproducibility
Disadvantages
•Large quantities of purified, high molecular weight DNA required (5-10 µg)
•Laborious and technically demanding
•High costs involved
•Distribution across the genome may be non-random
•Similar sized fragments may not be homologous (in case of multilocus probes)
•Difficulties in comparing polymorphisms across different gels
Obtaining a DNA fingerprint by using a VNTR
probe. (a) Preparation of the probe. The first
intron of the myoglobin gene has four repeats
of the sequence shown, which contains a 13bp core sequence (shown in boldface). This
core sequence is found at other VNTR loci,
labeled VNTR I, II, and III in this simple
diagrammatic representation. (b) The number
of repeats at the three VNTR loci with the
core sequence. The Southern blot has been
probed with the 33-bp repeat in part a and
shows the DNA fingerprints of three people.
Fig. 5 Détection des réarrangements du minisatellite CEB1-1.8 et CEB1-0.6
dans les cellules rad27Δ de S. cerevisiae. L'instabilité se manifeste par
l'apparition de variants de nouvelles tailles, plus courtes ou plus longues que
celles des allèles parentaux (indiqués par des flèches).
SNP
SNP (single nucleotide polymorphism
Is a DNA sequence variation occurring when a
single nucleotide — A, T, C, or G — in the genome
(or other shared sequence) differs between
members of a species (or between paired
chromosomes in an individual). For example, two
sequenced DNA fragments from different
individuals, AAGCCTA to AAGCTTA, contain a
difference in a single nucleotide. In this case we
say that there are two alleles : C and T. Almost all
common SNPs have only two alleles.
Variations in the DNA sequences of humans can
affect how humans develop diseases and respond
to pathogens, chemicals, drugs, vaccines, and
other agents. SNPs are also thought to be key
enablers in realizing the concept of personalized
medicine.[3] However, their greatest importance in
biomedical research is for comparing regions of
the genome between cohorts (such as with
matched cohorts with and without a disease).
The study of single-nucleotide polymorphisms is
also important in crop and livestock breeding
programs
Advantages and disadvantages of various DNA fingerprinting
methods
Type
Advantages
Disadvantages
Isozymes
Inexpensive
Co-dominant
Expressed genes
The small number of isozymes available can lead to
poor discrimination capacity between samples.
Poor resolution of electrophoretic bands. Markers can
be difficult to differentiate because of complex banding
patterns of oligomeric isozymes and co-migration in
electrophoresis
RAPD
Inexpensive
Simple
Uncharacterized DNA which is amplified in RAPD
provides no additional information, which is sometimes a
useful property of other marker systems. Low stringency
of the PCR process used in RAPD can result in profile
reproducibility problems.
Unknown gene function
AFLP
High discrimination
Many markers
Expensive
Complex procedure
Unknown gene function
SSR
High discrimination
Highly reproducible
Co-dominant
Characterized DNA
Complex procedure to establish initially for given taxa.
Expensive (unless using extant SSR primers). Taxaspecific, there are limitations to the transferability of
SSR markers between taxa; only successful with closely
related genera
Unknown gene function (unless EST-derived)
RFLP
Highly reproducible
Co-dominant
Characterized DNA
Complex procedure
Laborious
SNP
High discrimination
Co-dominant
Complex to establish initially for given taxa
Restriction site Associated DNA (RAD)
markers
Restriction site associated DNA (RAD) markers can be identified
and typed by detecting differential hybridization patterns of RAD
tags on a microarray. Genomic DNA samples S1 and S2 contain
the recognition sequence for various restriction enzymes at
locations throughout the genome. Dark blue triangles represent
restriction sites of a particular enzyme. Some of these restriction
sites are only present in one sample because of polymorphisms
that disrupt the recognition sequence (red asterisks). The two
samples are separately digested with a particular restriction
enzyme and then ligated to biotinylated linkers (light blue
ellipses). The DNA is randomly sheared leaving only the
fragments that were directly flanking a restriction site attached to
biotin linkers. These fragments are purified using streptavidin
beads and released by digestion at the original restriction site.
Loci containing polymorphisms, such as the left locus of S2 or
the right locus of S1, will not contain tags for that locus in the
purified RAD-tag sample, thus resulting in differential
hybridization patterns of RAD tags on a microarray.
Miller M R et al. Genome Res. 2007;17:240-248
Enriched RAD marker microarray
production and characterization
Enriched RAD marker microarray production and characterization.
RAD-tag samples S1 and S2 contain polymorphic sets of RAD tags. RAD
tags that are present in both individuals will not serve as informative
markers. In order to produce an array that types a large number of
informative markers, subtractive hybridization is used to enrich for
sample-specific RAD tags. RAD-tag clone libraries are generated from
these enriched samples. These clone libraries are used as templates for
PCR, the products of which are spotted to produce RAD marker
microarrays. To identify informative markers, RAD-tag samples S1 and
S2 are fluorescently labeled and competitively hybridized directly against
each other to the array.
Miller M R et al. Genome Res. 2007;17:240-248
The process of RADSeq
The process of RADSeq
(A) Genomic DNA is sheared with a restriction enzyme
of choice (SbfI in this example).
(B) P1 adapter is ligated to SbfI-cut fragments. The P1
adapter is adapted from the Illumina sequencing
adapter (full sequence not shown here), with a
molecular identifier (MID; CGATA in this example) and
a cut site overhang at the end (TGCA in this example).
(C) Samples from multiple individuals are pooled
together and all fragments are randomly sheared.
Only a subset of the resulting fragments contains
restriction sites and P1 adapters.
(D) P2 adapter is ligated to all fragments. The P2
adapter has a divergent end.
(E) PCR amplification with P1 and P2 primers. The P2
adapter will be completed only in the fragments ligated
with P1 adapter, and so only these fragments will be
fully amplified.
(F) Pooled samples with different MIDs are separated
bioinformatically and SNPs called (C/G SNP
underlined).
(G) As fragments are sheared randomly, paired end
sequences from each sequenced fragment will cover
a 300–400 bp region downstream of the restriction
site.
Davey J W , Blaxter M L Briefings in Functional Genomics 2010;9:416-423
Gene tagging
•
•
•
Transposon tagging
T-DNA tagging
Activation tagging
Activation TAGGING
Transposon tagging
TAIL-PCR
is very simple, efficient, and
highly specific. Since no other manipulations apart from PCR
are required, TAIL-PCR is especially suitable for isolation of
targeted unknown sequences from a large number of samples.
This technique has been used to recover insert ends from rice
BAC clones for chromosome walking and mapping.
AIMS (Amplification of insertion mutagenised sites)
Amplification of insertion
mutagenised sites (AIMS)
Reverse genetics
Reverse genetics is an approach to discovering the function of a gene that proceeds oppositely to
how such discoveries typically unfold in classical genetics, or in forward genetics.
By the classical approach, geneticists first look for rare individuals with unusual traits or
phenotypes, and then they trace these traits to an underlying faulty allele or gene. Locating the
gene on its chromosome is the end point of an investigation.
With the readily performed modern techniques of DNA sequencing and as a result of the
sequencing of many whole genomes, many genetic sequences are discovered in advance of any
other information about them. To learn the influence a sequence has on phenotype, or to discover
its biological function, researchers may engineer a:
•
•
•
Change or disruption by site-directed mutagenesis, for example, or by deletion of a gene by
gene knockout (as can be done in some organisms, such as yeast and mice) -and only
afterwards look for the effect of such alterations in the whole organism.
The discovery of gene silencing using double stranded RNA also known as RNA interference
has also made this approach very promising.
A third reverse genetics technique is the creation of transgenic organisms that overexpress a
gene of interest. The resulting phenotype may reflect the normal function of the gene.