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Chapter7 Analyzing DNA ＆gene structure,
variation ＆expression
 §1 sequencing ＆ genotyping DNA
 §2 Identifying genes in cloned DNA ＆ establishing their
structure
 §3 Studying gene expression
Ⅰ.sequencing ＆ genotyping DNA
 Chemical degradation method for DNA
sequencing (Maxam ＆Gillbert,1977)
 Enzymatic method for DNA sequencing (Fred
Sauger,1977)
◆The Enzymatic method, in which the sequence of a
single-stranded DNA molecule is determined by
enzymatic synthesis of complementary polynucleotide
chains, these chains terminating at specific nucleotide
positions
Enzymatic method for DNA sequencing
(Sanger Method / chain terminator sequencing)
principle :
Chain termination DNA sequencing is based on the
principle that single-stranded DNA molecules that differ in
length by just a single nucleotide can be separated from
one another by polyacrylamide gel electrophoresis .This
means that it is possible to resolve a family of molecules,
representing all lengths from 10 to 1500 nucleotides, into a
series of bands
ddCTP
why we use ddCTP?
dCTP
The polymerase enzyme does not discriminate between dNTPs and
ddNTPs, so the dideoxynucleotide can be incorporated into the growing
chain, but it then blocks further elongation because it lacks the 3hydroxyl group needed to form a connection with the next nucleotide
 We need the primer when sequencing.
why?
the primer is needed because template-dependent DNA
polymerases cannot initiate DNA synthesis on a
molecule that is entirely single-stranded: there must be a
short double-stranded region to provide a 3 - end onto
which the enzyme can add new nucleotides.
 Chain termination sequencing requires
a single-stranded DNA template
The template for a chain termination experiment is a
single-stranded version of the DNA molecule to be
sequenced.
 The DNA can be cloned in a plasmid vector
The resulting DNA will be double stranded so cannot be
used directly in sequencing. Instead, it must be
converted into single-stranded DNA by denaturation with
alkali or by boiling.
shortcoming :it can be difficult to prepare plasmid DNA
that is not contaminated with small quantities of bacterial
DNA and RNA, which can act as spurious templates or
primers in the DNA sequencing experiment.
 The DNA can be cloned in a bacteriophage
M13 vector.
Obtaining single-stranded DNA by cloning in a bacteriophage M13 vector

polymerase
Three criterion in particular must be fulfilled by a sequencing enzyme:
1.
High processivity
must have high processivity so that it does not
dissociate from the template before incorporating a
chain-terminating nucleotide.
1.
Negligible or zero 5 → 3 exonuclease activity
2.
Negligible or zero 3
→5 exonuclease activity
※desirable the polymerase does not remove the chain
termination nucleotide once it has been incorporated.
Kelenow enzyme; Taq polymerase; sequenase;
two advantages over traditional chain termination
sequencing
1.
2.
very little template DNA is needed, so the DNA does not have to
be cloned before being sequenced.
uses double-stranded rather than single-stranded DNA as the
starting material.
cycle sequencing
Automated DNA sequencing
Automated DNA sequencing using fluorescent primers
This method uses fluorescence labeling, the
DNA labeled by incorporating a primer or dNTP
which carries a fluorescence. Unlike
conventional DNA sequencing, this method use
of different fluorescence in the 4 base, and all 4
reactions can be loaded into a single lane.
During electrophoresis, a monitor detects and
records the fluorescence signal as the DNA
passes through a fixed point in the gel . The
output is in the form of intensity profiles for each
of the differently colored fluorophores , but the
information is simultaneously stored
electronically. This precludes transcription errors
when an interpreted sequence is typed by hand
into a computer file.
Ⅱ. Indentifying genes is cloned DNA ＆
establishing their structure
 Exon trapping
a technique for detecting sequences within a
cloned genomic DNA that are capable of splicing
to exons within a specialized vector.
This requires a special type of vector that contains a
minigene consisting of two exons flanking an intron
sequence, the first exon being preceded by the
sequence signals needed to initiate transcription in a
eukaryotic cell .To use the vector the piece of DNA to be
studied is inserted into a restriction site located within the
vector's intron region. The vector is then introduced into
a suitable eukaryotic cell line, where it is transcribed and
the RNA produced from it is spliced. The result is that
any exon contained in the genomic fragment becomes
attached between the upstream and downstream exons
from the minigene. RT-PCR with primers annealing
within the two minigene exons is now used to amplify a
DNA fragment, which is sequenced. As the minigene
sequence is already known, the nucleotide positions at
which the inserted exon starts and ends can be
determined, precisely delineating this exon.
cDNA selection/capture
a hybridization-based method for retrieving genomic clones
that have counterparts in a cDNA library
principle :
cognate cDNAs corresponding to genes found within the
YAC will bind preferentially to the YAC DNA.
*YAC: yeast artificial chromosome
early approaches used immobilized YACs and
NOW used solution hybridization reaction and
biotin- streptavidin capture methods
cDNA selection/capture
(magnetic bead )
RACE-PCR
(rapid amplification of cDNA ends-PCR)
A PCR-based technique for mapping the
end of an RNA molecule.
5`RACE-PCR
In the simplest form of this method one of the primers is
specific for an internal region close to the beginning of
the gene being studied. This primer attaches to the
mRNA for the gene and directs the first reversetranscriptase-catalyzed stage of the process, during
which a cDNA corresponding to the start of the mRNA is
made . Because only a small segment of the mRNA is
being copied, the expectation is that the cDNA synthesis
will not terminate prematurely, so one end of the cDNA
will correspond exactly with the start of the mRNA. Once
the cDNA has been made, a short poly(A) tail is attached
to its 3 - end. The second primer anneals to this poly(A)
sequence and, during the first round of the normal PCR,
converts the single-stranded cDNA into a doublestranded molecule, which is subsequently amplified as
the PCR proceeds.
Mapping transcription start and sites and
defining exon-intron boundaries
Nuclease S1 protection and primer extension
assay
 Primer extension assay
Primer extension is used to map the 5' ends of
DNA or RNA fragments. It is done by annealing
a specific oligonucleotide primer to a position
downstream of that 5' end. The primer is labeled,
usually at its 5' end, with 32P. This is extended
with reverse transcriptase, which can copy either
an RNA or a DNA template, making a fragment
that ends at the 5' end of the template molecule.
DNA polymerase can also be used with DNA
templates.
 Nuclease S1 protection
The 5`- or 3`-end of a transcript can be
identified by hybridization a longer, endlabeled antisense fragment to the RNA.
The hybrid is treated with nuclease S1 to
remove single-stranded regions, and the
remaining fragment`s size is measured on
a gel.
Ⅲ.Studying gene expression
Gene expression screening
↓
Target
↙
↘
RNA transcripts
protein
↓
↓
Hybridization
antibody
gene-based expression analyses:
hybridization analyses
1.Northern blot hybridization
2.Tissue in situ hybridization
3.Whole mount in situ hybridization
PCR analyses
1.RT-PCR
2.m RNA differential display
Ⅱ.protein-based expression analyses:

Immunoblotting (Western blotting)


Immunocytochemistry
→immunohistochemistry
Immunofluorescence microscopy

Ultrastructural studies
Northern blot hybridization
This approach affords low resolution
expression patterns by hybridizing a gene
or cDNA probe to total RNA or poly (A)+
RNA extracts prepared from different
tissues or cell lines. Because the RNA is
size-fractionated on a gel, it is possible to
estimate the size of transcripts. The
presence of multiple hybridization bands in
one lane may indicate the presence of
differently sized isoforms.
Tissue in situ hybridization
In situ hybridization (ISH) is a type of hybridization that
uses a labeled complementary DNA or RNA strand to
localize a specific DNA or RNA sequence in a portion or
section of tissue.
Whole mount in situ hybridization
an extension of tissue ISH is to study expression in a
whole embryo.
and whole mount ISH is a popular methods for tracking
expression during development in whole embryos from
vertebrate organisms.
※fluorescence in situ hybridization (FISH)
for single cell expression profiling
FISH is a cytogenetic technique which can be
used to detect and localize the presence or
absence of specific DNA sequences on
chromosomes. It uses fluorescent probes which
bind only to those parts of the chromosome with
which they show a high degree of sequence
similarity.
※large-scale expression screening using
microarrays
Chromosome FISH (fluorescence in situ hybridization)
mRNA differential display
a PCR-based technique for comparing the
mRNA species that are expressed in two
related sources of cells to pick out
differentially expressed genes.
the method uses a modified oligo(dT) primer which has a
different single nucleotide or dinucleotide at the 3- end
causing it to bind to the poly(A) tail of a subset of
mRNAs. For example, if the oligonucleotide
TTTTTTTTTTTCA(T11CA) is used as a primer, it will
preferentially prime cDNA synthesis from those mRNAs
where the dinucleotide TG precedes the poly(A) tail. The
second primer which is used is usually an arbitrary short
sequence (often 10 nucleotides long but, because of
mismatching, especially at the 5 - end, it can bind to
many more sites than expected for a decamer). The
resulting amplification patterns are deliberately designed
to produce a complex ladder of bands when sizefractionated in a long polyacrylamide gel .
Antibody labeling and detection system
the primary antibody is used as an
intermediate molecule and is not linked
directly to a labeled group. Once bound to
its target, the primary antibody is in turn
bound by a secondary reagent which is
conjugated to a reporter which may be a
fluorochrome, an enzyme or colloidal gold.
Antibody labeling-detection for tracking
protein expression
Immunoblotting (Western blotting)
a method to detect protein in a given sample of tissue
homogenate or extract.
first uses gel electrophoresis to separate denatured
proteins by mass. The proteins are then transferred out
of the gel and onto a membrane where they are "probed"
using antibodies specific to the protein. As a result, we
can examine the amount of protein in a given sample
and compare levels between several groups.
Immunohistochemistry (IHC)
Immunohistochemistry refers to the
process of localizing proteins in cells
of a tissue section exploiting the
principle of Abs binding specifically to
Ags in biological tissues.
Sometimes used to screen RNA
expression
immunocytochemistry
Immunofluorescence microscopy
This method is used when investigating
the subcellular location for a protein of
interest. A suitable fluorescent dye, such
as fluorescein or rhodamine is coupled to
the desired antibody, enabling the relevant
protein to be localized within the cell by
fluorescence microscopy
Ultrastructural studies
Higher resolution still of the intracellular
localization of a gene product or other
molecule is possible using electron
microscopy. The antibody is typically
labeled with an electron-dense particle,
such as colloidal gold spheres.
Green fluorescent protein (GFP)
the GFP gene is frequently used as a
reporter gene
when the GFP gene was cloned and
transfected into target cells in culture,
expression of GFP in heterologous cells
was also marked by emission of the green
fluorescent light.
GFP ribbon diagram
expression of GFP
in HeLa Cells
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