23-10-2018

Universidad de Chile

Programa Académico de Bachillerato

Eduardo Kessi C.
Departamento de Ciencias Biológicas Animales
Facultad de Ciencias Veterinarias y Pecuarias
Universidad de Chile
ekessi@uchile.cl

DNA Recombinante

EKC 2018 1
 23-10-2018

Some Major Steps in the Development of Recombinant

DNA and Transgenic Technology

1869 Miescher first isolates DNA from white blood cells harvested from pus-soaked
bandages obtained from a nearby hospital.
1944 Avery provides evidence that DNA, rather than protein, carries the genetic
information during bacterial transformation.
1953 Watson and Crick propose the double-helix model for DNA structure based on x-
ray results of Franklin and Wilkins.
1955 Kornberg discovers DNA polymerase, the enzyme now used to produce labeled
DNA probes.
1961 Marmur and Doty discover DNA renaturation, establishing the specificity and
feasibility of nucleic acid hydridization reactions.

1962 Arber provides the first evidence for the existence of DNA restriction nucleases,
leading to their purification and use in DNA sequence characterization by Nathans
and H. Smith.
1966 Nirenberg, Ochoa, and Khorana elucidate the genetic code.

1967 Gellert discovers DNA ligase, the enzyme used to join DNA fragments together.

Some Major Steps in the Development of Recombinant

DNA and Transgenic Technology
1972 DNA cloning techniques are developed by the laboratories of Boyer, Cohen, Berg,
1973 and their colleagues at Stanford University and the University of California at San
Francisco.
1975 Southern develops gel-transfer hybridization for the detection of specific DNA
sequences.
1975 Sanger and Barrell and Maxam and Gilbert develop rapid DNA-sequencing
1977 methods.
1981 Palmiter and Brinster produce transgenic mice; Spradling and Rubin produce
1982 transgenic fruit flies.
1982 GenBank, NIH's public genetic sequence database, is established at Los Alamos
National Laboratory.
1985 Mullis and co-workers invent the polymerase chain reaction (PCR).

1987 Capecchi and Smithies introduce methods for performing targeted gene replacement
in mouse embryonic stem cells.
1989 Fields and Song develop the yeast two-hybrid system for identifying and studying
protein interactions
1989 Olson and colleagues describe sequence-tagged sites, unique stretches of DNA that
are used to make physical maps of human chromosomes.

EKC 2018 2
 23-10-2018

Some Major Steps in the Development of Recombinant

DNA and Transgenic Technology

1990 Lipman and colleagues release BLAST, an algorithm used to search for homology
between DNA and protein sequences.
1990 Simon and colleagues study how to efficiently use bacterial artificial chromosomes,
BACs, to carry large pieces of cloned human DNA for sequencing.
1991 Hood and Hunkapillar introduce new automated DNA sequence technology.
1995 Venter and colleagues sequence the first complete genome, that of the bacterium
Haemophilus influenzae.
1996 Goffeau and an international consortium of researchers announce the completion of
the first genome sequence of a eucaryote, the yeast Saccharomyces cerevisiae.
1996 Lockhart and colleagues and Brown and DeRisi produce DNA microarrays, which
1997 allow the simultaneous monitoring of thousands of genes.
1998 Sulston and Waterston and colleagues produce the first complete sequence of a
multicellular organism, the nematode worm Caenorhabditis elegans.
2001 Consortia of researchers announce the completion of the draft human genome
sequence.

EKC 2018 3
 23-10-2018

Un Organismo Genéticamente Modificado (GMO- Genetically Modified Organism)

es cualquier organismo cuyo material genético se ha modificado (alterado) mediante el
uso de técnicas de ingeniería genética

Organismo genéticamente modificado Aquel organismo que ha sufrido algún

grado de modificación de su genoma (introducción, supresión o multiplicación de genes
de la misma especie o la introducción de genes de especies diferentes), por
métodos generalmente artificiales.

Transgénico es un organismo animal o vegetal, perteneciente a una determinada especie

biológica, que ha incorporado de manera estable uno o más genes de otro organismo, de
una especie biológica diferente, y lo hereda a sus descendientes

Transgén es un gen o material genético que se ha transferido de forma natural, o por

cualquiera de las técnicas de ingeniería genética de un organismo a otro. La
introducción de un transgén tiene el potencial de cambiar el fenotipo del organismo
receptor. En su uso más preciso, el término transgén describe un segmento de ADN que
contiene una secuencia del gen que se ha aislado de un organismo y se introduce en un
organismo diferente

Los genes de eucariontes generalmente se clonan (amplifican) y secuencian en

hospedadores bacterianos, pero se introducen en un organismo eucarionte, ya sea la
especie donante original o una diferente. El gen transferido se llama un transgén, y el
producto de la ingeniería genética se llama un organismo transgénico

Clonación de DNA

1.- Cortar el DNA de manera precisa (repetitiva)

2.- Seleccionar una molécula de DNA pequeña capaz de

experimentar replicación (vector)

3.- Juntar (ligar) covalentemente fragmentos de DNA

4.- Poner la molécula de DNA recombinante en una célula

hospedera (amplificación, mantención)

5.- Identificar (y seleccionar) las células hospederas que

contienen la molécula de DNA recombinante

EKC 2018 4
 23-10-2018

Use of pBR322 to clone and identify foreign DNA in E. coli.

The insertion of a DNA fragment into a bacterial plasmid with the enzyme DNA
ligase. The plasmid is cut open with a restriction nuclease (in this case, one that
produces staggered ends) and is mixed with the DNA fragment to be cloned (which has
been prepared with the same restriction nuclease). DNA ligase and ATP are added. The
staggered ends basepair, and DNA ligase seals the nicks in the DNA backbone,
producing a complete recombinant DNA molecule. In the accompanying micrographs,
the inserted DNA is colored red

EKC 2018 5
 23-10-2018

Algunas enzimas usadas para producir DNA recombinante

Enzima(s) Función
Endonucleasas de restricción II Cortan el DNA reconociendo secuencias específicas

DNA ligasa Unen covalentemente dos fragmentos de DNA

DNA polimerasa I (E. coli) Llenan gaps en dsDNA adicionando nucleótidos al

extremo 3’OH
Trancriptasa inversa Produce DNA usando un molde de RNA

Polinucleótido quinasa Agrega un fosforilo al extremo 5`OH de polinucleótido

para marcarlo o permitir ligarlo

Transferasa terminal Agrega colas homopliméricas al extremo 3’OH de un

dsDNA lineal

Exonucleasa III Elimina nucleótidos del extremo 3`OH de una hebra

de DNA

 exonucleasa Elimina nucleótidos de los extremos 5`de un dsDNA

para exponer extremos 3’OH de hebra simple

Fosfata alcalina Elimina fosoforilos del extremo 5’ o 3´ (o ambos)

Síntesis de Proinsulina humana en

una bacteria. Proinsulina, el
Transcriptasa precursor de la insulina, puede
inversa sintetizarse en clones transformados
(genéticamente alterados) de E. coli.
Los clones contienen (y en este caso
expresan, el gen de la proinsulina
humana.
Pancreas mRNA de cDNA de
proinsulina proinsulina
humana

Gen para proinsulina

proinsulina
Ligar al Transformar
plasmidio E. coli

cDNA de Plasmidio Bacteria

proinsulina recombinante transformante

EKC 2018 6
 23-10-2018

Enzimas de restricción y separación

de moléculas (fragmentos) de DNA

Secuencias de reconocimiento para algunas endonucleasas de restricción tipo II

Las flechas indican los enlaces que corta cada enzima. Los asteriscos indican las bases que son metiladas
por la metilasa correspondiente (cuando se conoce). N indica cualquiera de las bases. El nombre de una
enzima consiste de una abreviatura de tres (en cursiva) del nombre de la especie bacteriana de la cual se
obtiene seguida, a veces, por el nombre de la cepa y números romanos para indicarlas diferentes enzimas
aisladas desde la misma especie de bacteria. Por ejemplo, BamHI es la primera (I) endonucleasa de
restricción que se aisló de Bacillus amyloliquefaciens, cepa H.

EKC 2018 7
 23-10-2018

The DNA nucleotide sequences recognized by four

widely used restriction nucleases. As in the
examples shown, such sequences are often six base
pairs long and "palindromic" (that is, the nucleotide
sequence is the same if the helix is turned by 180
degrees around the center of the short region of helix
that is recognized). The enzymes cut the two strands
of DNA at or near the recognition sequence. For
some enzymes, such as HpaI, the cleavage leaves
blunt ends; for others, such as EcoRI, HindIII, and
PstI, the cleavage is staggered and creates cohesive
ends. Restriction nucleases are obtained from various
species of bacteria: HpaI is from Hemophilus
parainfluenzae, EcoRI is from Escherichia coli,
HindIII is from Hemophilus influenzae, and PstI is
from Providencia stuartii.

EKC 2018 8
 23-10-2018

Restriction nucleases produce DNA

fragments that can be easily joined
together. Fragments with the same
cohesive ends can readily join by
complementary base-pairing between
their cohesive ends, as illustrated. The
two DNA fragments that join in this
example were both produced by the
EcoRI restriction nuclease, whereas the
three other fragments were produced
by different restriction nucleases that
generated different cohesive ends

Cleavage of DNA molecules by restriction endonucleases. Restriction endonucleases

recognize and cleave only specific sequences,leaving either (a) sticky ends or (b) blunt
ends. Fragments can be ligated to other DNAs, such as the cleaved cloning vector shown
here. This reaction is facilitated by the annealing of complementary sticky ends. Ligation
is less efficient for DNA fragments with blunt ends.

EKC 2018 9
 23-10-2018

A synthetic DNA fragment with recognition sequences for several restriction endonucleases
can be inserted into a plasmid that has been cleaved by a restriction endonuclease. The
insert is called a linker; an insert with multiple restriction sites is called a polylinker.

EKC 2018 10
 23-10-2018

Vectores de clonamiento

Plasmidios

Bacteriófagos

BACs

YACs

The constructed E. coli plasmid pBR322. Note the location of some important restriction
sites—for PstI, EcoRI, BamHI, SalI, and PvuII; ampicillin- and tetracycline-resistance genes;
and the replication origin (ori). Constructed in 1977, this was one of the early plasmids
designed expressly for cloning in E. coli.

EKC 2018 11
 23-10-2018

The restriction enzyme EcoRI cuts a circular DNA molecule bearing one target
sequence, resulting in a linear molecule with single-stranded sticky ends.

Some of the different ways of introducing foreign DNA into a cell

EKC 2018 12
 23-10-2018

1. pBR322 has an origin of replication, ori, a

sequence where replication is initiated by cellular
enzymes. This sequence is required to propagate the
plasmid and maintain it at a level of 10 to 20 copies
per cell.
2. The plasmid contains two genes that confer
resistance to different antibiotics (tetR, ampR),
allowing the identification of cells that contain the
intact plasmid or a recombinant version of the
plasmid.
3. Several unique recognition sequences in pBR322
(PstI, EcoRI, BamHI, SalI, PvuII) are targets for
different restriction endonucleases, providing sites
where the plasmid can later be cut to insert foreign
DNA.
4. The small size of the plasmid (4,361 bp) facilitates
its entry into cells and the biochemical manipulation
of DNA. The entry is achieved by transformation (in
a CaCl containing medium) or electroporation.
Useful fragment size for plasmids are up to 15 Kbp

Bacteriophage cloning vectors.

Recombinant DNA methods are
used to modify the bacteriophage
genome, removing the genes not
needed for phage production and
replacing them with “filler” DNA to
make the phage DNA large enough
for packaging into phage particles.
As shown here, the filler is replaced
with foreign DNA in cloning
experiments. Recombinants are
packaged into viable phage particles
in vitro only if they include an
appropriately sized foreign DNA
fragment as well as both of the
essential DNA end fragments.

EKC 2018 13
 23-10-2018

Bacteriophage has a very efficient mechanism for delivering its

48,502 bp of DNA into a bacterium, and it can be used as a vector to
clone somewhat larger DNA segments. Two key features contribute
to its utility:
1. About one-third of the  genome is nonessential and can be
replaced with foreign DNA.
2. DNA is packaged into infectious phage particles only if it is
between 40 and 53 Kbp long, a constraint that can be used to ensure
packaging of recombinant DNA only. Bacteriophage  vectors permit
the cloning of DNA fragments of up to 23 Kbp. Transformation of E.
coli is higly efficient.

Bacterial artificial chromosomes (BACs) as

cloning vectors. The vector is a relatively
simple plasmid, with a replication origin (ori).
The par genes, derived from a type of plasmid
called an F plasmid, assist in the even
distribution of plasmids to daughter cells at cell
division. The BAC includes selectable markers.
A lacZ gene is situated in the cloning region
such that it is inactivated by cloned DNA
inserts. Introduction of recombinant BACs into
cells by electroporation is promoted by the use
of cells with an altered (more porous) cell wall.
Recombinant DNAs are screened for resistance
to the antibiotic chloramphenicol (CmR). Plates
also contain a substrate for –galactosidase that
yields a colored product. Colonies with active -
galactosidase and hence no DNA insert in the
BAC vector turn blue; colonies without -
galactosidase activity—and thus with the
desired DNA inserts—are white. These cloning
vectors are designed for the cloning of very
long segments (typically 100 to 300 Kbp) of
DNA

EKC 2018 14
 23-10-2018

Construction of a yeast artificial

chromosome (YAC). A YAC vector
includes an origin of replication (ori), a
centromere (CEN), two telomeres (TEL),
and selectable markers (X and Y). Digestion
with BamH1 and EcoRI generates two
separate DNA arms, each with a telomeric
end and one selectable marker. A large
segment of DNA (e.g., up to 2 x106 bp from
the human genome) is ligated to the two
arms to create a yeast artificial
chromosome. The YAC transforms yeast
cells (prepared by removal of the cell wall
to form spheroplasts), and the cells are
selected for X and Y; the surviving cells
propagate the DNA insert.

Sizes of inserted DNA commonly obtained with different cloning vectors

Cloning vector Size of insert

Standard high copy number plasmid vectors 0 10 kb

Bacteriophage  insertion vectors 0 10 kb
Bacteriophage  replacement vectors 9 23 kb
Cosmid vectors 30 44 kb
Bacteriophage P1 70 100 kb
PAC (P1 artificial chromosome) vectors 130 150 kb
BAC (bacterial artificial chromosome) vectors up to 300 kb
YAC (yeast artificial chromosome) vectors 0.2 2.0 Mb

EKC 2018 15
 23-10-2018

Use of hybridization to identify a clone

with a particular DNA segment. The
radioactive DNA probe hybridizes to
complementary DNA and is revealed by
autoradiography. Once the labeled colonies
have been identified, the corresponding
colonies on the original agar plate can be
used as a source of cloned DNA for further
study.

Probe to detect the gene for a protein of known amino acid sequence. Because more than
one DNA sequence can code for any given amino acid sequence, the genetic code is said to
be “degenerate.” Thus the correct DNA sequence for a known amino acid sequence cannot be
known in advance. The probe is designed to be complementary to a region of the gene with
minimal degeneracy, that is, a region with the fewest possible codons for the amino acids—
two codons at most in the example shown here. Oligonucleotides are synthesized with
selectively randomized sequences, so that they contain either of the two possible nucleotides
at each position of potential degeneracy (shaded in pink). The oligonucleotide shown here
represents a mixture of eight different sequences: one of the eight will complement the gene
perfectly, and all eight will match at least 17 of the 20 positions.

EKC 2018 16
 23-10-2018

Genotecas (librerías genómicas)

Construction of a human genomic DNA

library. A genomic library is usually stored as a
set of bacteria, each carrying a different
fragment of human DNA. For simplicity, cloning
of just a few representative fragments (colored)
is shown. In reality, all of the gray DNA
fragments would also be cloned.

EKC 2018 17
 23-10-2018

A DNA library is a collection of DNA

clones, gathered together as a source of
DNA for sequencing, gene discovery, or
gene function studies. The library can take a
variety of forms, depending on the source of
the DNA.

Among the largest types of DNA library is

a genomic library, produced when the
complete genome of a particular organism
is cleaved into thousands of fragments, and
all the fragments are cloned by insertion
into a cloning vector.

Preparación de una librería genómica

1) Digestión parcial del DNA genómico con endonucleasas de restricción de

modo que una secuencia cualquiera aparecerá en fragmentos de varios
tamaños.

2) Elegir un vector compatible con el tamaño de los fragmentos producidos,

lo que asegura que virtualmente todas las secuencias estarán representadas
en la librería.

3) Eliminar, por centrifugación o electroforesis, los fragmentos muy grandes

y los muy pequeños.

4) Tratar el vector (por ejemplo BAC o YAC) con la misma enzima de

restricción con que obtuvo los fragmentos de DNA genómico

5) Ligar los fragmentos y el vector mediante DNA ligasa

6) Transformar bacterias, o levaduras, de modo de producir una librería de

tipos de células que contienen una molécula específica, y única, de DNA
recombinante.

EKC 2018 18
 23-10-2018

Ordering of the clones in a DNA library. A segment of a chromosome from a

hypothetical organism X, with markers A-Q representing sequence-tagged sites (STSs—
DNA segments of known sequence). Below the chromosome is an array of ordered BAC
clones, numbered 1 to 9. The presence or absence of an STS on an individual clone can be
determined by hybridization—for example, by probing each clone with PCR-amplified
DNA from the STS. Once the STSs on each BAC clone are identified, the clones can be
ordered on the map. For example, compare clones 3, 4, and 5. Marker E (blue) is found on
all three clones; F (red) on clones 4 and 5, but not on 3; and G (green) only on clone 5.
This indicates that the order of the sites is E, F, G. The clones partially overlap and their
order must be 3, 4, 5. The resulting ordered series of clones is called a contig.

Comparison between the genetic and

physical maps of Saccharomyces
cerevisiae chromosome III. The
comparison shows the discrepancies
between the genetic and physical maps,
the latter determined by DNA
sequencing. Note that the order of the
upper two markers (glk1 and cha1) is
incorrect on the genetic map, and that
there are also differences in the relative
positioning of other pairs of markers.

EKC 2018 19
 23-10-2018

The synthesis of cDNA. Total

mRNA is extracted from a
particular tissue (brain in this
example), and DNA copies
(cDNA) of the mRNA
molecules are produced by the
enzyme reverse transcriptase
For simplicity, the copying of
just one of these mRNAs into
cDNA is illustrated.

The differences between cDNA clones and genomic DNA clones derived from the same
region of DNA. In this example gene A is infrequently transcribed, whereas gene B is
frequently transcribed, and both genes contain introns (green). In the genomic DNA library,
both the introns and the nontranscribed DNA (pink) are included in the clones, and most
clones contain, at most, only part of the coding sequence of a gene (red). In the cDNA clones
the intron sequences (yellow) have been removed by RNA splicing during the formation of the
mRNA (blue), and a continuous coding sequence is therefore present in each clone.

EKC 2018 20
 23-10-2018

The use of nucleic acid

hybridization to determine the
region of a cloned DNA fragment
that is present in an mRNA
molecule. The method shown
requires a nuclease that cuts the DNA
chain only where it is not base-paired
to a complementary RNA chain. The
positions of the introns in eucaryotic
genes are mapped by the method
shown; the beginning and the end of
an RNA molecule can be determined
in the same way. For this type of
analysis the DNA is electrophoresed
through a denaturing agarose gel,
which causes it to migrate as single-
stranded molecules.

Vectores de expresión

EKC 2018 21
 23-10-2018

DNA sequences in a typical E.

coli expression vector. The gene
to be expressed is inserted into one
of the restriction sites in the
polylinker, near the promoter (P),
with the end encoding the amino
terminus proximal to the promoter.
The promoter allows efficient
transcription of the inserted gene,
and the transcription termination
sequence sometimes improves the
amount and stability of the mRNA
produced. The operator (O)
permits regulation by means of a
repressor that binds to it. The
ribosome binding site provides
sequence signals needed for
efficient translation of the mRNA
derived from the gene. The
selectable marker allows the
selection of cells containing the
recombinant DNA.

Production of large amounts of a

protein from a protein-coding DNA
sequence cloned into an expression
vector and introduced into cells. A
plasmid vector has been engineered to
contain a highly active promoter, which
causes unusually large amounts of mRNA
to be produced from an adjacent protein-
coding gene inserted into the plasmid
vector. Depending on the characteristics
of the cloning vector, the plasmid is
introduced into bacterial, yeast, insect, or
mammalian cells, where the inserted gene
is efficiently transcribed and translated
into protein

EKC 2018 22
 23-10-2018

Síntesis de Proinsulina humana en

una bacteria. Proinsulina, el
Transcriptasa precursor de la insulina, puede
inversa sintetizarse en clones transformados
(genéticamente alterados) de E. coli.
Los clones contienen (y en este caso
expresan) el gen de la proinsulina
humana.
Pancreas mRNA de cDNA de
proinsulina proinsulina
humana

Gen para proinsulina

proinsulina
Ligar al Transformar
plasmidio E. coli

cDNA de Plasmidio Bacteria

proinsulina recombinante transformante

Production of large amounts of a protein

by using a plasmid expression vector. In
this example, bacterial cells have been
transfected with the coding sequence for an
enzyme, DNA helicase; transcription from
this coding sequence is under the control of
a viral promoter that becomes active only at
temperatures of 37°C or higher. The total
cell protein has been analyzed by SDS-
polyacrylamide gel electrophoresis, either
from bacteria grown at 25°C (no helicase
protein made), or after a shift of the same
bacteria to 42°C for up to 2 hours (helicase
protein has become the most abundant
protein species in the lysate).

EKC 2018 23
 23-10-2018

Síntesis de oligonucleótidos

Secuenciacón del DNA

PCR

EKC 2018 24
 23-10-2018

Basic thecniques in molecular biology

!. Restriction-enzyme analysis. Restriction enzymes are precise, molecular

scalpels that allow the investigator to manipulate DNA segments.

2. Blotting techniques. The Southern and Northern blots are used to separate
and characterize DNA and RNA, respectively.

3. DNA sequencing. The precise nucleotide sequence of a molecule of DNA can

be determined. Sequencing has yielded a wealth of information concerning gene
architecture, the control of gene expression, and protein structure.

4. Solid-phase synthesis of nucleic acids. Precise sequences of nucleic acids

can be synthesized de novo and used to identify or amplify other nucleic acids.

5. The polymerase chain reaction (PCR). The polymerase chain reaction leads
to a billionfold amplification of a segment of DNA. One molecule of DNA can
be amplified to quantities that permit characterization and manipulation. This
powerful technique is being used to detect pathogens and genetic diseases, to
determine the source of a hair left at the scene of a crime, and to resurrect genes
from fossils.

General approaches for studying specific DNA sequences in complex DNA

populations.

EKC 2018 25
 23-10-2018

Solid-Phase Synthesis of a DNA Chain by the Phosphite Triester Method. The

activated monomer added to the growing chain is a deoxyribonucleoside 3’-
phosphoramidite containing a DMT protecting group on its 5’ oxygen atom, a -
cyanoethyl (CE) protecting group on its 3’ phosphoryl oxygen, and a protecting
group on the base.

EKC 2018 26
 23-10-2018

EKC 2018 27
 23-10-2018

EKC 2018 28
 23-10-2018

Approaches to site-directed mutagenesis. A

synthetic oligonucleotide with a desired sequence
change at one position is hybridized to a single-
stranded copy of the gene to be altered. This acts as
primer for synthesis of a duplex DNA (with one
mismatch), which is then used to transform cells.
Cellular DNA repair systems will convert about 50%
of the mismatches to reflect the desired sequence
change.

The enzymatic or dideoxymethod of sequencing DNA. (A) This method relies on the use of
dideoxyribonucleoside triphosphates, derivatives of the normal deoxyribonucleoside
triphosphates that lack the 3 hydroxyl group. (B) Purified DNA is synthesized in vitro in a
mixture that contains single-stranded molecules of the DNA to be sequenced (gray), the enzyme
DNA polymerase, a short primer DNA (orange) to enable the polymerase to start DNA synthesis
(C) To determine the complete sequence of a DNA fragment, the double-stranded DNA is first
separated into its single strands and one of the strands is used as the template for sequencing.

EKC 2018 29
 23-10-2018

Desnaturación para separar

las hebras de DNA

EKC 2018 30
 23-10-2018

Fluorescence Detection of Oligonucleotide Fragments Produced by the Dideoxy

Method. Each of the four chain-terminating mixtures is primed with a tag that
fluoresces at a different wavelength (e.g., blue for A). The sequence determined by
fluorescence measurements at four wavelengths is shown at the bottom

EKC 2018 31
 23-10-2018

Automated DNA sequencing. Shown here is a tiny part of the data from an
automated DNA-sequencing run as it appears on the computer screen. Each colored
peak represents a nucleotide in the DNA sequencea clear stretch of nucleotide
sequence can be read here between positions 173 and 194 from the start of the
sequence. This particular example is taken from the international project that
determined the complete nucleotide sequence of the genome of the plant
Arabidopsis.

A Complete Genome. The diagram

depicts the genome of Haemophilus
influenzae, the first complete
genome of a free-living organism to
be sequenced. The genome encodes
more than 1700 proteins and 70
RNA molecules. The likely function
of approximately one-half of the
proteins was determined by
comparisons with sequences from
proteins previously characterized in
other species

EKC 2018 32
 23-10-2018

The First Cycle in the

Polymerase Chain Reaction
(PCR). A cycle consists of
three steps: strand separation,
hybridization of primers, and
extension of primers by DNA
synthesis

EKC 2018 33
 23-10-2018

Use of PCR to obtain a genomic or cDNA

clone. (A) To obtain a genomic clone by
using PCR, chromosomal DNA is first
purified from cells. PCR primers that flank
the stretch of DNA to be cloned are added,
and many cycles of the reaction are
completed. Since only the DNA between
(and including) the primers is amplified,
PCR provides a way to obtain a short
stretch of chromosomal DNA selectively in
a pure form. (B) To use PCR to obtain a
cDNA clone of a gene, mRNA is first
purified from cells. The first primer is then
added to the population of mRNAs, and
reverse transcriptase is used to make a
complementary DNA strand. The second
primer is then added, and the single-
stranded DNA molecule is amplified
through many cycles of PCR, as shown in.
For both types of cloning, the nucleotide
sequence of at least part of the region to be
cloned must be known beforehand.

EKC 2018 34
 23-10-2018

Generating a restriction map. The size patterns from double digests provide
information on the relative locations of restriction sites. The example shows
size fractionation by agarose gel electrophoresis of restriction fragments
following incubation of a 6.2 kb DNA fragment with the indicated enzymes.
New bands in the double digests (i.e. not found in the original single digests)
are indicated by black boxes

Transcriptome analysis. (A) Transcriptome

analysis with a DNA chip carrying
oligonucleotides representing all the genes in a
small genome. After adding labeled cDNA,
the positions of the hybridization signals on
the chip indicate which genes have contributed
to the transcriptome under study. (B) With a
larger genome, cDNA clones prepared from
the transcriptome of one tissue are
immobilized as a microarray and probed with
cDNAs representing the same or a different
transcriptome. By comparing the hybridization
patterns, genes that are expressed differently
in the tissues from which the transcriptomes
are obtained can be identified.

EKC 2018 35
 23-10-2018

DNA microarray. A microarray can be prepared

from any known DNA sequence, from any source,
generated by chemical synthesis or by PCR. The
DNA is positioned on a solid surface (usually
specially treated glass slides) with the aid of a
robotic device capable of depositing very small
(nanoliter) drops in precise patterns. UV light
cross-links the DNA to the glass slides. Once the
DNA is attached to the surface, the microarray can
be probed with other fluorescently labeled nucleic
acids. Here, mRNA samples are collected from
cells at two different stages in the development of
a frog. The cDNA probes are made with
nucleotides that fluoresce in different colors for
each sample; a mixture of the cDNAs is used to
probe the microarray. Green spots represent
mRNAs more abundant at the single-cell stage;
red spots, sequences more abundant later in
development. The yellow spots indicate
approximately equal abundance at both stages.

EKC 2018 36
 23-10-2018

Enlarged image of a DNA microarray. Each glowing spot in this microarray contains DNA
from one of the 6,200 genes of the yeast (S. cerevisiae) genome, with every gene represented
in the array. The microarray has been probed with fluorescently labeled nucleic acid derived
from the mRNAs obtained (1) when the cells were growing normally in culture and (2) five
hours after the cells began to form spores. The green spots represent genes expressed at higher
levels during normal growth; the red spots, genes expressed at higher levels during sporulation.
The yellow spots represent genes that do not change their levels of expression during
sporulation. This image is enlarged; the microarray actually measures only 1.8 1.8 cm.

DNA of higher eukaryotes consists of unique and repeated sequences. Only 1.5
percent of human DNA encodes proteins and functional RNAs and the regulatory
sequences that control their expression

EKC 2018 37
 23-10-2018

Generación de repeticiones de microsatélites por deslizamiento hacia

atrás durante la replicación del DNA (un tipo de mutación)
Replicación normal

Deslizamiento hacia atrás

n puede variar típicamente entre 5 y 50

Segunda replicación

Segunda replicación
DNA “hijo”con una repetición extra

Estas dos variantes ocupan el

mismo locus en un par de
DNA “hijo” normal cromosomas homólogos, por tanto
son alelos

EKC 2018 38
 23-10-2018

¿Cuántos alelos puede haber por locus en una población determinada?

5’ CAG CAG CAG CAG CAG 3’

A5
3’ CAG CAG CAG CAG CAG 5’

5’ CAG CAG CAG CAG CAG CAG CAG 3’

A7
3’ CAG CAG CAG CAG CAG CAG CAG 5’

5’ CAG CAG CAG CAG CAG CAG CAG CAG CAG 3’

A9
3’ CAG CAG CAG CAG CAG CAG CAG CAG CAG 5’

5’ CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG 3’
A11
3’ CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG 5’

¿Cuántos genotipos se pueden predecir para estos cuatro alelos?

¿Si existieran sólo 12 loci, cuál sería el número de genotipos posibles?
¿Es verdadero que las frecuencias de los alelos son iguales entre distintas
poblaciones de individuos?
Terrier chileno presenta 17 loci con entre 4-14 alelos

Amplificar PCR

Separar Electroforesis

EKC 2018 39
 23-10-2018

Amplificación (clonamiento) in vitro de DNA mediante la reacción

de la polimerasa en cadena (PCR) (Poymerase Chain Reaction)

EKC 2018 40
 23-10-2018

Mezcla de
moléculas
de DNA

Electroforesis

Dirección
de la
electroforesis Gel poroso

Electroforesis en gel. (A). Típicamente, varias muestras se someten a electroforesis en un

gel plano (de poliacrilamida o de agarosa). Se usa una micropipeta para cargar las muestras
en cada pocillo. La cámara se cierra y se aplica corriente continua, generalmente a una
diferencia de potencial fija. Las moléculas cargadas negativamente migran en dirección
del polo positivo, en la parte de abajo del gel. (B) El efecto de tamizaje del gel poroso
separa las moléculas de acuerdo con su tamaño, de modo que las moléculas más pequeñas
se mueven más rápido.

Determinación Identificación en
de paternidad escena de crimen
Sospechosos
Especimen
Víctima

EKC 2018 41
 23-10-2018

Loci usados en Chile para identificación genética en Homo sapiens.

Sistema CODIS (Combined DNA Index System)

EKC 2018 42
 23-10-2018

Gene therapy is an experimental technique for treating disease by altering

the patient's genetic material. Most often, gene therapy works by introducing
a healthy copy of a defective gene into the patient's cells.

EKC 2018 43
 23-10-2018

Figure 7–78 CRISPR-mediated immunity in bacteria and archaebacteria. After infection by a virus
(left panel), a small bit of DNA from the viral genome is inserted into the CRISPR locus. For this
to happen, a small fraction of infected cells must survive the initial viral infection. The surviving
cells, or more generally their descendants, transcribe the CRISPR locus and process the transcript
into crRNAs (middle panel). Upon reinfection with a virus that the population has already been
“vaccinated” against, the incoming viral DNA is destroyed by a complementary crRNA (right
panel). For a CRISPR system to be effective, the crRNAs must not destroy the CRISPR locus
itself, even though the crRNAs are complementary in sequence to it. In many species, in order for
crRNAs to attack an invading DNA molecule, there must be additional short nucleotide sequences
that are carried by the target molecule. Because these sequences, known as PAMs (protospacer
adjacent motifs), lie outside the crRNA sequences, the host CRISPR locus is spared (see Figure 8–
55).

Leford H .2016 Nature 431: 156-152

EKC 2018 44