Welcome Each of
You to My
Molecular Biology
Class
2
Molecular Biology of the Gene,
5/E --- Watson et al,(2004)
Part I,Chemistry and Genetics
Part II,Maintenance of the Genome
Part III,Expression of the Genome
Part IV,Regulation
Part V,Methods
3
Ch 20,Techniques of
Molecular Biology
Ch 21,Model Organisms
Part V,METHODS
4
?Molecular Biology Course
Chapter 20
Techniques of
Molecular Biology
Preparation,analysis and manipulation
of nucleic acids and proteins
5
The methods depend upon,and were
developed from,an understanding
of the properties of biological
macromolecules themselves.
? Hybridization---the base-pairing
characteristics of DNA and RNA
? DNA cloning--- DNA polymerase,
restriction endonucleases and DNA
ligase
? PCR---Thermophilic DNA polymerase
6
Topic1,Nucleic acids
CHAPTER20,Techniques of Molecular Biology
1.Electrophoresis
2.Restriction
3.Hybridization
4.DNA Cloning and gene expression
5.PCR
6.Genome sequence & analysis
7
1,Gel electrophoresis separates
DNA and RNA molecules
according to size,shape and
topological properties
Gel matrix is an inserted,jello-like
porous material that support and
allows macromolecules to move
through,Agarose and polyacrylamide
are two different gel matrices
Electrophoresis
8
? DNA and RNA molecules are
negatively charged,thus move in the
gel matrix toward the positive pole (+)
? Linear DNA molecules are separated
according to size
? The mobility of circular DNA
molecules is affected by their
topological structures,The mobility
of the same molecular weight DNA
molecule with different shapes is,
supercoiled> linear> nicked or relaxed
Electrophoresis
9
Fig 20-1,DNA separation by gel electrophoresis
large moderate small After electr
10
To separate DNA of different
size ranges
? Narrow size range of DNA,use
polyacrylamide
? Wide size range of DNA,use
agarose gel
? Very large DNA(>30-50kb),use
pulsed-field gel electrophoresis
Electrophoresis
11
pulsed-field gel
electrophoresis
Switching between two
orientations,the larger the
DNA is,the longer it takes
to reorient
Electrophoresis
12
Restriction endonucleases
cleave DNA molecules at
particular sites
Nu
cle
ic
a
cid
?Why use endonucleases?
--To make large DNA molecules
break into manageable
fragments
Restriction digestion
13
?Restriction endonucleases,
the nucleases that cleave DNA at
particular sites by the
recognition of specific sequences
? The target site recognized by
endonucleases is usually
palindromic (回文结构 ),
e.g,EcoRI 5’…,GAATTC.…,3’
…,CTTAAG…,
Restriction digestion
14
?To name a restriction
endonuclease,
e.g,EcoRI
the 1st such
enzyme foundEscherichia coli
Species category
R13
strain
Restriction digestion
15
?Frequency of the
occurrence of hexamaeric
sequence,
1/4096 (4-6)
Randomly
Restriction digestion
16
? Consider a linear DNA
molecule with 6 copies
of GAATTC,
it will be cut into 7
fragments which could
be separated in the gel
electrophoresis by size
(The largest fragment) (The smallest
fragment)
Fig 20-3 digestionof a DNA fragment with endonuclease EcoRI
17
? Endonucleases are used to make
restriction map:
? e.g,the combination of EcoRI + HindIII
? Allows different regions of one molecule
to be isolate and a given molecule to be
identified
? A given molecule will generate a
characteristic series of patterns when
digested with a set of different enzymes
Restriction digestion
18
Different enzymes recognize their
specific target sites with different
frequency
? EcoRI Recognize hexameric sequence,4-6
? Sau3A1 Recognize terameric sequence,4-
4
? Thus Sau3A1 cuts the same DNA
molecule more frequently
Restriction digestion
19
sticky ends
blunt ends
Fig 20-4 recognition sequences and cut sites of various endonucleases
Restriction digestion
20
? The 5’ protruding ends of are said to be
“sticky” because they readily anneal through
base-pairing to DNA molecules cut with the
same enzyme
Reanneal with its
complementary
strand or other
strands with the
same cut
Restriction digestion
21
DNA hybridization can be
used to identify specific
DNA molecules
Nu
cle
ic
a
cid
Hybridization,the process of
base-pairing between
complementary ssDNA or RNA
from two different sources
DNA hybridization
22
?Probe,a labeled,defined
sequence used to search
mixtures of nucleic acids for
molecules containing a
complementary sequence
Labeling of DNA or RNA probes
End labeling,put the labels at the
ends
Uniform labeling,put the labels
internally
radioactive labeling,display and/or magnify
the signals by radioactivity
Non-radioactive labeling,display and/or
magnify the signals by antigen labeling – antibody
binding – enzyme binding - substrate application
(signal release)
End labeling
Single stranded DNA/RNA
5’ -end labeling,polynucleotide
kinase (PNK)
3’-end labeling,terminal
transferase
Labeling at both ends by kinase,
then remove one end by
restriction digestion
---------------------G
---------------------CTTAAp5’
5’ pAATTC
G
Uniformly labeling of DNA/RNA
Nick translation:
DNase I to introduce random nicks
?DNA polI to remove dNMPs from 3’ to 5’
and add new dNMP including labeled
nucleotide at the 3’ ends.
Hexanucleotide primered labeling:
Denature DNA ? add random
hexanucleotide primers and DNA pol ?
synthesis of new strand incorporating
labeled nucleotide,
J1 Characterization of clones
Strand-specific DNA probes:
e.g,M13 DNA as template
the missing strand can be re-
synthesized by incorporating
radioactive nulceotides
Strand-specific RNA probes:
labeled by transcription
J1 Characterization of clones
J1-5 Southern and Northern blotting
DNA on blot RNA on blot
1,Genomic DNA preparation RNA preparation
2,Restriction digestion -
3,Denature with alkali -
4,Agarose gel electrophoresis ?
5,DNA blotting/transfer and fixation RNA
6,Probe labeling ?
6,Hybridization (temperature) ?
7,Signal detection (X-ray film or antibody) ?
J1 Characterization of clones
Southern analysis
31
Southern bolt
hybridization
bI1 bI2 bI3 bI4 bI5
Northern analysis COB RNAs in S,cerevisiae
mRNA
Pre-mRNAs
Blot type Target Probe Applications
Southern DNA DNA or
RNA
mapping genomic
clones
estimating gene
numbers
Northern RNA DNA or
RNA
RNA sizes,
abundance,
and expression
Western Protei
n
Antibodi
es
protein size,
abundance
J1 Characterization of clones
34
Sequencing
Nu
cle
ic
a
cid
Two ways for sequencing:
?1,DNA molecules
(radioactively labeled at 5’
termini) are subjected to 4
regiments to be broken
preferentially at Gs,Cs,Ts,
As,separately.
?2,chain-termination method
35
chain-termination method
? ddNTPs are chain-terminating nucleotides,
the synthesis of a DNA strand stops when a
ddNTP is added to the 3’ end
36
The absence of 3’-hydroxyl lead to the
inefficiency of the nucleophilic attack on
the next incoming substrate molecule
37
DNA synthesis aborts
at a frequency of 1/100
every time the
polymerase meets a
ddGTP
Tell from the gel the
position of each G
38
Fig 20-15 DNA sequencing gel
4 systems with dNTP+
ddGTP,dNTP+ ddATP d
NTP+ ddCTP,d NTP+
ddTTP separately
“read” the sequencing
gel to get the
sequence of the DNA
39
The shortgun strategy permits
a partial assembly of large
genome sequence
? If we want to sequence a much larger
and more complicate eukaryotic
genome using the shortgun strategy,
What can we do?
? Firstly,libraries in different level
should be constructed.
NU
CL
EI
C
AC
ID
S
40Fig 20-16
41
? The DNA fragment can be easily
extracted and sequenced automatically.
? Sophisticated computer programs have
been developed to assemble the
randomized DNA fragment,forming
contigs.
? A single contig is about 50000 to
200000 bp,It’s useful to analysis fruit
fly genome that contains an average of
one gene every 10kb.
? If we want to analysis human genome,
contigs should be assembled into
scaffolds,
42
1-16 the paired-end strategy
permits the assembly of large
genome sequence
? The main limitation to producing large
contigs is the occurrence of repetitive
sequence,(Why?)
? To solve this problem,paired-end
sequencing is developed.
? The same genomic DNA is also used
to produce recombinant libraries
composed of large fragments between
3~100kb.
NU
CL
EI
C
AC
ID
S
43
? The end of each clone can be sequenced
easily,and these larger clones can firstly
assemble together,
44
? If a larger scaffold is needed,you
should use a cloning vector that can
carry large DNA fragment,(at least
100kb),BAC is a good choice,
45
1-17 genome-wide analysis
? The purpose of this analysis is to
predict the coding sequence and
other functional sequence in the
genome.
? For the genomes of bacteria and
simple eukaryotes,finding ORF is
very simple and effective.
NU
CL
EI
C
AC
ID
S
46
? For animal genomes,a variety of
bioinformatics tools are required to
identify genes and other functional
fragments,But the accuracy is low,
Fig 20-18
47
? The most important method for validating
protein coding regions and identify those
those missed by current current gene
finder program is the use of cDNA
sequence data.
? The mRNAs are firstly reverse transcript
into cDNA,and these cDNA,both full
length and partial,are sequenced using
shortgun method,These sequence are
used to generate EST (expressed
sequence tag) database,And these
ESTs are aligned onto genomic scaffolds
to help us identify genes.
48
Part II proteins
49
2-1 specific proteins can be
purified from cell extracts
? The purification of individual proteins is
critical to understanding their function,
(why?)
? Although there are thousands of
proteins in a single cell,each protein
has unique properties that make its
purification somewhat different from
others.
pr
ot
ein
s
50
? The purification of a protein is
designed to exploit its unique
characteristics,such as size,
charge,shape,and in many
instance,function.
51
2-2 purification of a protein
requires a specific assay
? To purify a protein requires that you
have an assay that is unique to that
protein.
? In many instance,it’s convenient to
use a measure for the function of the
protein,or you may use the antibody
of the protein.
? It is useful to monitor the purification
process.
pr
ot
ein
s
52
2-4 Proteins can be separated
from one another using
column chromatography
? In this approach,protein fractions
are passed though glass columns
filled with appropriated modified
small acrylamide or agarose beads.
? There are various ways columns can
be used to separate proteins
according to their characteristics,
pr
ot
ein
s
53
Ion exchange chromatography
? The proteins are separated according
to their surface charge.
? The beads are modified with either
negative-charged or positive-charged
chemical groups.
? Proteins bind more strongly requires
more salt to be eluted.
54
Fig 20-22-a
55
Gel filtration
chromatography
? This technique separate the proteins on
the bases of size and shape,
? The beads for it have a variety of
different sized pores throughout.
? Small proteins can enter all of the pores,
and take longer to elute; but large
proteins pass quickly.
56
Fig 20-22-b
57
2-5 affinity chromatography can
facilitate more rapid protein
purification
? If we firstly know our target protein can
specifically interact with something else,
we can bind this,something else” to
the column and only our target protein
bind to the column.
This method is called affinity
chromatography,
pr
ot
ein
s
58
Immunoaffinity
chromatography
? An antibody that is specific for the
target is attached to the bead,and
ideally only the target protein can
bind to the column.
? However,sometimes the binding is
too tight to elute our target protein,
unless it is denatured,But the
denatured protein is useless.
59
? Sometimes tags (epitopes) can be
added to the N- or C- terminal of the
protein,using molecular cloning method.
? This procedure allows the modified
proteins to be purified using
immunoaffinity purification and a
heterologous antibody to the tag.
? Importantly,the binding affinity can
change according to the condition,e.g,
the concentration of the Ca2+ in the
solution.
60
immunoprecipitation
? We attach the antibody to the bead,
and use it to precipitate a specific
protein from a crude cell extract.
? It’s a useful method to detect what
proteins or other molecules are
associated with the target protein,
61
2-6 separation of proteins
on polyacrylamide gels
? The native proteins have neither a
uniform charge nor a uniform
secondary structure.
? If we treat the protein with a strong
detergent SDS,the higher structure is
usually eliminated,And SDS confers
the polypeptide chain a uniform
negative charge.
pr
ot
ein
s
62
? And sometimes mercaptoethanol is
need to break the disulphide bond.
? Thus,the protein molecules can be
resolved by electrophoresis in the
presence of SDS according to the
length of individual polypeptide.
? After electrophoresis,the proteins can
be visualized with a stain,such as
Coomassie brilliant blue.
63
2-7 antibodies visualize
electrophoretically-separated
proteins,
? The electrophoretically separated
proteins are transferred to a filter,And
this filter is then incubate in a solution
of an antibody to our interested protein,
Finally,a chromogenic enzyme is used
to visualized the filter-bound antibody
pr
ot
ein
s
64
2-8 protein molecules can
be directly sequenced
? Two sequence method,Edman
degradation & Tandem mass
spectrometry(MS/MS).
? Due to the vast resource of
complete or nearly complete
genome,the determination of even
a small stretch of protein sequence
is sufficient to identify the gene.
pr
ot
ein
s
65
Edman degradation
? It’s a chemical reaction in which the
amino acid’s residues are
sequentially release for the N-
terminus of a polypeptide chain.
66
? Step 1,modify the N-terminal amino
with PITC,which can only react with
the free α-amino group.
? Step 2,cleave off the N-terminal by
acid treatment,but the rest of the
polypeptide remains intact.
? Step 3,identify the released amino
acids by High Performance Liquid
Chromatography (HPLC).
The whole process can be carried out in
an automatic protein sequencer,
67
Fig 20-23
68
Tandem mass spectrometry
? MS is a method in which the mass
of very small samples of a
material can be determined.
69
? Step 1,digest your target protein into
short peptide.
? Step 2,subject the mixture of the
peptide to MS,and each individual
peptide will be separate.
? Step 3,capture the individual peptide
and fragmented into all the component
peptide.
? Step 4,determine the mass of each
component peptide.
? Step 5:Deconvolution of these data and
the sequence will be revealed,
70
Fig 20-24
71
2-9 proteomics
? Proteomics is concerned with the
identification of the full set of
proteins produced by a cell or a
tissue under a particular by a
particular set of conditions,
pr
ot
ein
s
72
Three principle methods
? 1,2-D gel electrophoresis for protein
separation.
? 2,MS for the precise determination of a
protein.
? 3,Bioinformatics technology.
73
1-14 shortgun sequencing a
bacterial genome
? The bacterium H,influenzae was the
first free-living organism to have a
complete genome sequenced and
assembled.
? This organism is chosen as its
genome is small (1.8Mb) and
compact,
NU
CL
EI
C
AC
ID
S
74
? Its whole genome was sheared into many
random fragments with an average
length of 1kb.
? This pieces are cloned into a plasmid
vector,And these clones are sequenced
respectively.
? All these sequence information are loaded
into the computer,The powerful program
will assemble the random DNA fragment
based on containing matching sequence,
forming a single continuous assemble,
called a contig,
75
? To ensure every nucleotide in the
genome was captured in the final
genome assemble,30000~40000 clones
are needed,which is ten times larger as
the genome,This is called
10× sequence coverage.
? This method might seem tedious,but it’s
much faster and cheaper than the
digestion-mapping-sequencing method,
As the computer is much faster at
assembling sequence than the time
required to map the chromosome,
J3 Polymerase chain reaction
J3-1 PCR
J3-2 The PCR cycle
J3-3 Template
J3-4 Primers
J3-5 Enzymes
J3-6 PCR optimization
Analysis and uses of cloned DNA
J3-1 PCR
The polymerase chain
reaction(PCR) is to used to
amplify a sequence of DNA
using a pair of primers each
complementary to one end
of the the DNA target
sequence.
J3 Polymerase chain reaction
J3-2 The PCR cycle
? Denaturation,The target DNA
(template) is separated into two stands
by heating to 95℃
? Primer annealing,The temperature is
reduced to around 55℃ to allow the
primers to anneal.
? Polymerization (elongation,
extension),The temperature is increased
to 72℃ for optimal polymerization step
which uses up dNTPs and required Mg++.
J3 Polymerase chain reaction
J3 Polymerase chain reaction
Template
Primers
Enzymes
Fig,
Steps of PCR
J2 nucleic acid sequencing
J3-3 Template
?Any source of DNA that provides
one or more target molecules can
in principle be used as a template
for PCR
?Whatever the source of template
DNA,PCR can only be applied if
some sequence information is
known so that primers can be
designed.
J3 Polymerase chain reaction
J3-4 Primers
? PCR primers need to be about 18 to 30
nt long and have similar G+C contents
so that they anneal to their
complementary sequences at similar
temperatures.They are designed to
anneal on opposite strands of the
target sequence.
? Tm=2(a+t)+4(g+c),determine
annealing temperature,If the primer is
18-30 nt,annealing temperature can be
Tm?5oC
J3 Polymerase chain reaction
Degenerate primers,an oligo
pool derived from protein sequence.
E.g,His-Phe-Pro-Phe-Met-Lys can
generate a primer
5’-CAY TTY CCN TTY ATG AAR
Y= Pyrimidine
N= any base
R= purine
J3 Polymerase chain reaction
J3-5&6 Enzymes and PCR
Optimization
? The most common is Taq
polymerase.It has no 3’ to 5’
proofreading exonuclease activity,
Accuracy is low,not good for
cloning.
? We can change the annealing
temperature and the Mg++
concentration or carry out nested
PCR to optimize PCR.
J3 Polymerase chain reaction
PCR optimization
I.Reverse transcriptase-PCR
II.Nested PCR
J2 nucleic acid sequencing
Fig Nested PCR
First roundprimers
First roundPCR
Second roundprimers
Second round
PCR
Gene of interest
J2 nucleic acid sequencing
Reverse transcriptase-PCR
AAA(A)n
5‘ -Cap mRNA
(dT)12~18
primer anneal5‘ -Cap
AAA(A)n
3‘ 5‘
Reverse transcriptasedNTP
5‘ -Cap
AAA(A)n
5‘
cDNA:mRNA hybrid
Regular
PCR
Fig
RT-PCR
J2 nucleic acid sequencing
You to My
Molecular Biology
Class
2
Molecular Biology of the Gene,
5/E --- Watson et al,(2004)
Part I,Chemistry and Genetics
Part II,Maintenance of the Genome
Part III,Expression of the Genome
Part IV,Regulation
Part V,Methods
3
Ch 20,Techniques of
Molecular Biology
Ch 21,Model Organisms
Part V,METHODS
4
?Molecular Biology Course
Chapter 20
Techniques of
Molecular Biology
Preparation,analysis and manipulation
of nucleic acids and proteins
5
The methods depend upon,and were
developed from,an understanding
of the properties of biological
macromolecules themselves.
? Hybridization---the base-pairing
characteristics of DNA and RNA
? DNA cloning--- DNA polymerase,
restriction endonucleases and DNA
ligase
? PCR---Thermophilic DNA polymerase
6
Topic1,Nucleic acids
CHAPTER20,Techniques of Molecular Biology
1.Electrophoresis
2.Restriction
3.Hybridization
4.DNA Cloning and gene expression
5.PCR
6.Genome sequence & analysis
7
1,Gel electrophoresis separates
DNA and RNA molecules
according to size,shape and
topological properties
Gel matrix is an inserted,jello-like
porous material that support and
allows macromolecules to move
through,Agarose and polyacrylamide
are two different gel matrices
Electrophoresis
8
? DNA and RNA molecules are
negatively charged,thus move in the
gel matrix toward the positive pole (+)
? Linear DNA molecules are separated
according to size
? The mobility of circular DNA
molecules is affected by their
topological structures,The mobility
of the same molecular weight DNA
molecule with different shapes is,
supercoiled> linear> nicked or relaxed
Electrophoresis
9
Fig 20-1,DNA separation by gel electrophoresis
large moderate small After electr
10
To separate DNA of different
size ranges
? Narrow size range of DNA,use
polyacrylamide
? Wide size range of DNA,use
agarose gel
? Very large DNA(>30-50kb),use
pulsed-field gel electrophoresis
Electrophoresis
11
pulsed-field gel
electrophoresis
Switching between two
orientations,the larger the
DNA is,the longer it takes
to reorient
Electrophoresis
12
Restriction endonucleases
cleave DNA molecules at
particular sites
Nu
cle
ic
a
cid
?Why use endonucleases?
--To make large DNA molecules
break into manageable
fragments
Restriction digestion
13
?Restriction endonucleases,
the nucleases that cleave DNA at
particular sites by the
recognition of specific sequences
? The target site recognized by
endonucleases is usually
palindromic (回文结构 ),
e.g,EcoRI 5’…,GAATTC.…,3’
…,CTTAAG…,
Restriction digestion
14
?To name a restriction
endonuclease,
e.g,EcoRI
the 1st such
enzyme foundEscherichia coli
Species category
R13
strain
Restriction digestion
15
?Frequency of the
occurrence of hexamaeric
sequence,
1/4096 (4-6)
Randomly
Restriction digestion
16
? Consider a linear DNA
molecule with 6 copies
of GAATTC,
it will be cut into 7
fragments which could
be separated in the gel
electrophoresis by size
(The largest fragment) (The smallest
fragment)
Fig 20-3 digestionof a DNA fragment with endonuclease EcoRI
17
? Endonucleases are used to make
restriction map:
? e.g,the combination of EcoRI + HindIII
? Allows different regions of one molecule
to be isolate and a given molecule to be
identified
? A given molecule will generate a
characteristic series of patterns when
digested with a set of different enzymes
Restriction digestion
18
Different enzymes recognize their
specific target sites with different
frequency
? EcoRI Recognize hexameric sequence,4-6
? Sau3A1 Recognize terameric sequence,4-
4
? Thus Sau3A1 cuts the same DNA
molecule more frequently
Restriction digestion
19
sticky ends
blunt ends
Fig 20-4 recognition sequences and cut sites of various endonucleases
Restriction digestion
20
? The 5’ protruding ends of are said to be
“sticky” because they readily anneal through
base-pairing to DNA molecules cut with the
same enzyme
Reanneal with its
complementary
strand or other
strands with the
same cut
Restriction digestion
21
DNA hybridization can be
used to identify specific
DNA molecules
Nu
cle
ic
a
cid
Hybridization,the process of
base-pairing between
complementary ssDNA or RNA
from two different sources
DNA hybridization
22
?Probe,a labeled,defined
sequence used to search
mixtures of nucleic acids for
molecules containing a
complementary sequence
Labeling of DNA or RNA probes
End labeling,put the labels at the
ends
Uniform labeling,put the labels
internally
radioactive labeling,display and/or magnify
the signals by radioactivity
Non-radioactive labeling,display and/or
magnify the signals by antigen labeling – antibody
binding – enzyme binding - substrate application
(signal release)
End labeling
Single stranded DNA/RNA
5’ -end labeling,polynucleotide
kinase (PNK)
3’-end labeling,terminal
transferase
Labeling at both ends by kinase,
then remove one end by
restriction digestion
---------------------G
---------------------CTTAAp5’
5’ pAATTC
G
Uniformly labeling of DNA/RNA
Nick translation:
DNase I to introduce random nicks
?DNA polI to remove dNMPs from 3’ to 5’
and add new dNMP including labeled
nucleotide at the 3’ ends.
Hexanucleotide primered labeling:
Denature DNA ? add random
hexanucleotide primers and DNA pol ?
synthesis of new strand incorporating
labeled nucleotide,
J1 Characterization of clones
Strand-specific DNA probes:
e.g,M13 DNA as template
the missing strand can be re-
synthesized by incorporating
radioactive nulceotides
Strand-specific RNA probes:
labeled by transcription
J1 Characterization of clones
J1-5 Southern and Northern blotting
DNA on blot RNA on blot
1,Genomic DNA preparation RNA preparation
2,Restriction digestion -
3,Denature with alkali -
4,Agarose gel electrophoresis ?
5,DNA blotting/transfer and fixation RNA
6,Probe labeling ?
6,Hybridization (temperature) ?
7,Signal detection (X-ray film or antibody) ?
J1 Characterization of clones
Southern analysis
31
Southern bolt
hybridization
bI1 bI2 bI3 bI4 bI5
Northern analysis COB RNAs in S,cerevisiae
mRNA
Pre-mRNAs
Blot type Target Probe Applications
Southern DNA DNA or
RNA
mapping genomic
clones
estimating gene
numbers
Northern RNA DNA or
RNA
RNA sizes,
abundance,
and expression
Western Protei
n
Antibodi
es
protein size,
abundance
J1 Characterization of clones
34
Sequencing
Nu
cle
ic
a
cid
Two ways for sequencing:
?1,DNA molecules
(radioactively labeled at 5’
termini) are subjected to 4
regiments to be broken
preferentially at Gs,Cs,Ts,
As,separately.
?2,chain-termination method
35
chain-termination method
? ddNTPs are chain-terminating nucleotides,
the synthesis of a DNA strand stops when a
ddNTP is added to the 3’ end
36
The absence of 3’-hydroxyl lead to the
inefficiency of the nucleophilic attack on
the next incoming substrate molecule
37
DNA synthesis aborts
at a frequency of 1/100
every time the
polymerase meets a
ddGTP
Tell from the gel the
position of each G
38
Fig 20-15 DNA sequencing gel
4 systems with dNTP+
ddGTP,dNTP+ ddATP d
NTP+ ddCTP,d NTP+
ddTTP separately
“read” the sequencing
gel to get the
sequence of the DNA
39
The shortgun strategy permits
a partial assembly of large
genome sequence
? If we want to sequence a much larger
and more complicate eukaryotic
genome using the shortgun strategy,
What can we do?
? Firstly,libraries in different level
should be constructed.
NU
CL
EI
C
AC
ID
S
40Fig 20-16
41
? The DNA fragment can be easily
extracted and sequenced automatically.
? Sophisticated computer programs have
been developed to assemble the
randomized DNA fragment,forming
contigs.
? A single contig is about 50000 to
200000 bp,It’s useful to analysis fruit
fly genome that contains an average of
one gene every 10kb.
? If we want to analysis human genome,
contigs should be assembled into
scaffolds,
42
1-16 the paired-end strategy
permits the assembly of large
genome sequence
? The main limitation to producing large
contigs is the occurrence of repetitive
sequence,(Why?)
? To solve this problem,paired-end
sequencing is developed.
? The same genomic DNA is also used
to produce recombinant libraries
composed of large fragments between
3~100kb.
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? The end of each clone can be sequenced
easily,and these larger clones can firstly
assemble together,
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? If a larger scaffold is needed,you
should use a cloning vector that can
carry large DNA fragment,(at least
100kb),BAC is a good choice,
45
1-17 genome-wide analysis
? The purpose of this analysis is to
predict the coding sequence and
other functional sequence in the
genome.
? For the genomes of bacteria and
simple eukaryotes,finding ORF is
very simple and effective.
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? For animal genomes,a variety of
bioinformatics tools are required to
identify genes and other functional
fragments,But the accuracy is low,
Fig 20-18
47
? The most important method for validating
protein coding regions and identify those
those missed by current current gene
finder program is the use of cDNA
sequence data.
? The mRNAs are firstly reverse transcript
into cDNA,and these cDNA,both full
length and partial,are sequenced using
shortgun method,These sequence are
used to generate EST (expressed
sequence tag) database,And these
ESTs are aligned onto genomic scaffolds
to help us identify genes.
48
Part II proteins
49
2-1 specific proteins can be
purified from cell extracts
? The purification of individual proteins is
critical to understanding their function,
(why?)
? Although there are thousands of
proteins in a single cell,each protein
has unique properties that make its
purification somewhat different from
others.
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? The purification of a protein is
designed to exploit its unique
characteristics,such as size,
charge,shape,and in many
instance,function.
51
2-2 purification of a protein
requires a specific assay
? To purify a protein requires that you
have an assay that is unique to that
protein.
? In many instance,it’s convenient to
use a measure for the function of the
protein,or you may use the antibody
of the protein.
? It is useful to monitor the purification
process.
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2-4 Proteins can be separated
from one another using
column chromatography
? In this approach,protein fractions
are passed though glass columns
filled with appropriated modified
small acrylamide or agarose beads.
? There are various ways columns can
be used to separate proteins
according to their characteristics,
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Ion exchange chromatography
? The proteins are separated according
to their surface charge.
? The beads are modified with either
negative-charged or positive-charged
chemical groups.
? Proteins bind more strongly requires
more salt to be eluted.
54
Fig 20-22-a
55
Gel filtration
chromatography
? This technique separate the proteins on
the bases of size and shape,
? The beads for it have a variety of
different sized pores throughout.
? Small proteins can enter all of the pores,
and take longer to elute; but large
proteins pass quickly.
56
Fig 20-22-b
57
2-5 affinity chromatography can
facilitate more rapid protein
purification
? If we firstly know our target protein can
specifically interact with something else,
we can bind this,something else” to
the column and only our target protein
bind to the column.
This method is called affinity
chromatography,
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Immunoaffinity
chromatography
? An antibody that is specific for the
target is attached to the bead,and
ideally only the target protein can
bind to the column.
? However,sometimes the binding is
too tight to elute our target protein,
unless it is denatured,But the
denatured protein is useless.
59
? Sometimes tags (epitopes) can be
added to the N- or C- terminal of the
protein,using molecular cloning method.
? This procedure allows the modified
proteins to be purified using
immunoaffinity purification and a
heterologous antibody to the tag.
? Importantly,the binding affinity can
change according to the condition,e.g,
the concentration of the Ca2+ in the
solution.
60
immunoprecipitation
? We attach the antibody to the bead,
and use it to precipitate a specific
protein from a crude cell extract.
? It’s a useful method to detect what
proteins or other molecules are
associated with the target protein,
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2-6 separation of proteins
on polyacrylamide gels
? The native proteins have neither a
uniform charge nor a uniform
secondary structure.
? If we treat the protein with a strong
detergent SDS,the higher structure is
usually eliminated,And SDS confers
the polypeptide chain a uniform
negative charge.
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? And sometimes mercaptoethanol is
need to break the disulphide bond.
? Thus,the protein molecules can be
resolved by electrophoresis in the
presence of SDS according to the
length of individual polypeptide.
? After electrophoresis,the proteins can
be visualized with a stain,such as
Coomassie brilliant blue.
63
2-7 antibodies visualize
electrophoretically-separated
proteins,
? The electrophoretically separated
proteins are transferred to a filter,And
this filter is then incubate in a solution
of an antibody to our interested protein,
Finally,a chromogenic enzyme is used
to visualized the filter-bound antibody
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2-8 protein molecules can
be directly sequenced
? Two sequence method,Edman
degradation & Tandem mass
spectrometry(MS/MS).
? Due to the vast resource of
complete or nearly complete
genome,the determination of even
a small stretch of protein sequence
is sufficient to identify the gene.
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Edman degradation
? It’s a chemical reaction in which the
amino acid’s residues are
sequentially release for the N-
terminus of a polypeptide chain.
66
? Step 1,modify the N-terminal amino
with PITC,which can only react with
the free α-amino group.
? Step 2,cleave off the N-terminal by
acid treatment,but the rest of the
polypeptide remains intact.
? Step 3,identify the released amino
acids by High Performance Liquid
Chromatography (HPLC).
The whole process can be carried out in
an automatic protein sequencer,
67
Fig 20-23
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Tandem mass spectrometry
? MS is a method in which the mass
of very small samples of a
material can be determined.
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? Step 1,digest your target protein into
short peptide.
? Step 2,subject the mixture of the
peptide to MS,and each individual
peptide will be separate.
? Step 3,capture the individual peptide
and fragmented into all the component
peptide.
? Step 4,determine the mass of each
component peptide.
? Step 5:Deconvolution of these data and
the sequence will be revealed,
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Fig 20-24
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2-9 proteomics
? Proteomics is concerned with the
identification of the full set of
proteins produced by a cell or a
tissue under a particular by a
particular set of conditions,
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Three principle methods
? 1,2-D gel electrophoresis for protein
separation.
? 2,MS for the precise determination of a
protein.
? 3,Bioinformatics technology.
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1-14 shortgun sequencing a
bacterial genome
? The bacterium H,influenzae was the
first free-living organism to have a
complete genome sequenced and
assembled.
? This organism is chosen as its
genome is small (1.8Mb) and
compact,
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? Its whole genome was sheared into many
random fragments with an average
length of 1kb.
? This pieces are cloned into a plasmid
vector,And these clones are sequenced
respectively.
? All these sequence information are loaded
into the computer,The powerful program
will assemble the random DNA fragment
based on containing matching sequence,
forming a single continuous assemble,
called a contig,
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? To ensure every nucleotide in the
genome was captured in the final
genome assemble,30000~40000 clones
are needed,which is ten times larger as
the genome,This is called
10× sequence coverage.
? This method might seem tedious,but it’s
much faster and cheaper than the
digestion-mapping-sequencing method,
As the computer is much faster at
assembling sequence than the time
required to map the chromosome,
J3 Polymerase chain reaction
J3-1 PCR
J3-2 The PCR cycle
J3-3 Template
J3-4 Primers
J3-5 Enzymes
J3-6 PCR optimization
Analysis and uses of cloned DNA
J3-1 PCR
The polymerase chain
reaction(PCR) is to used to
amplify a sequence of DNA
using a pair of primers each
complementary to one end
of the the DNA target
sequence.
J3 Polymerase chain reaction
J3-2 The PCR cycle
? Denaturation,The target DNA
(template) is separated into two stands
by heating to 95℃
? Primer annealing,The temperature is
reduced to around 55℃ to allow the
primers to anneal.
? Polymerization (elongation,
extension),The temperature is increased
to 72℃ for optimal polymerization step
which uses up dNTPs and required Mg++.
J3 Polymerase chain reaction
J3 Polymerase chain reaction
Template
Primers
Enzymes
Fig,
Steps of PCR
J2 nucleic acid sequencing
J3-3 Template
?Any source of DNA that provides
one or more target molecules can
in principle be used as a template
for PCR
?Whatever the source of template
DNA,PCR can only be applied if
some sequence information is
known so that primers can be
designed.
J3 Polymerase chain reaction
J3-4 Primers
? PCR primers need to be about 18 to 30
nt long and have similar G+C contents
so that they anneal to their
complementary sequences at similar
temperatures.They are designed to
anneal on opposite strands of the
target sequence.
? Tm=2(a+t)+4(g+c),determine
annealing temperature,If the primer is
18-30 nt,annealing temperature can be
Tm?5oC
J3 Polymerase chain reaction
Degenerate primers,an oligo
pool derived from protein sequence.
E.g,His-Phe-Pro-Phe-Met-Lys can
generate a primer
5’-CAY TTY CCN TTY ATG AAR
Y= Pyrimidine
N= any base
R= purine
J3 Polymerase chain reaction
J3-5&6 Enzymes and PCR
Optimization
? The most common is Taq
polymerase.It has no 3’ to 5’
proofreading exonuclease activity,
Accuracy is low,not good for
cloning.
? We can change the annealing
temperature and the Mg++
concentration or carry out nested
PCR to optimize PCR.
J3 Polymerase chain reaction
PCR optimization
I.Reverse transcriptase-PCR
II.Nested PCR
J2 nucleic acid sequencing
Fig Nested PCR
First roundprimers
First roundPCR
Second roundprimers
Second round
PCR
Gene of interest
J2 nucleic acid sequencing
Reverse transcriptase-PCR
AAA(A)n
5‘ -Cap mRNA
(dT)12~18
primer anneal5‘ -Cap
AAA(A)n
3‘ 5‘
Reverse transcriptasedNTP
5‘ -Cap
AAA(A)n
5‘
cDNA:mRNA hybrid
Regular
PCR
Fig
RT-PCR
J2 nucleic acid sequencing
