Welcome to MB Class

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Welcome to MB Class
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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
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Part V METHODS

• Ch 20 Techniques of
• Molecular Biology
• Ch 21 Model Organisms

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• Molecular Biology Course

• Chapter 20
• Techniques of
• Molecular Biology

Preparation, analysis and manipulation of nucleic
acids and proteins
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• 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

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CHAPTER20 Techniques of Molecular Biology

• Topic 1 Nucleic acids

1. Separation by Electrophoresis (????)
2. Cut by Restriction endonuclease (????????)
3. Identification by Hybridization (????)
4. Amplified by PCR (PCR??)
5. DNA Cloning and gene expression (DNA???????)
6. Genome sequence analysis (????????)

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1. Gel electrophoresis separates DNA and RNA
molecules according to size, shape and
topological properties
Gel Electrophoresis (????)
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DNA gel mobility (DNA???????)

• 1.DNA and RNA molecules are negatively charged,
  thus move in the gel matrix (????) toward the
  positive pole (???).
• 2.Linear DNA molecules are separated according to
  sizes. The large DNA molecules move slower than
  the small molecules.
• 3.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 (???)gt linear (??) gt nicked or
  relaxed (?????)

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DNA can be visualized by staining the gel with
fluorescent dyes, such as ethidium bromide (EB
????)
moderate
large
small
Fig 20-1 DNA is separated by gel electrophoresis
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Gel matrix (????)
Gel matrix (????) is an inserted, jello-like
porous material that supports and allows
macromolecules to move through.
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• Polyacrylamide (?????)
• has high resolving capability, and can resolve
  DNA that differ from each other as little as a
  single base pair/nucleotide.
• but can only separate DNA over a narrow size
  range (1 to a few hundred bp).

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• Agarose (???)
• a much less resolving power than polyacrylamide,
• but can separate DNA molecules of up to tens of kb

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Pulsed-field gel electrophoresis (????)

1. The electric field is applied in pulses that are
   oriented orthogonally (???) to each other.
2. Separate DNA molecules according to their
   molecule weight, as well as to their shape and
   topological properties.
3. Can effectively separate DNA molecules over 30-50
   kb and up to several Mb in length.

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Fig. 20-2 pulsed-field gel electrophoresis
Switching between two orientations the larger
the DNA is, the longer it takes to reorient
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Electrophoresis is also used to separate RNAs

1. RNA have a uniform negative charge as DNA does.
2. RNA is single-stranded and have extensive
   secondary and tertiary structure, which
   significantly influences their electrophoretic
   mobility.
3. RNA can be treated with reagent such as glyoxal
   (???) to prevent RNA base pairing, so that its
   mobility correlates with the molecular weight

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CHAPTER20 Techniques of Molecular Biology

• Topic 1 Nucleic acids

1. Separation by Electrophoresis (????)
2. Cut by Restriction endonuclease (????????)
3. Identification by Hybridization (????)
4. Amplified by PCR (PCR??)
5. DNA Cloning and gene expression (DNA???????)
6. Genome sequence analysis (????????)

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2. Restriction endonucleases (??????) cleave DNA
molecules at particular sites
Nucleic acids-Restriction digestion

• Why use endonucleases?
• --To break large DNA molecules into manageable
  fragments.

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• Restriction endonucleases (RE) are the nucleases
  that cleave DNA at particular sites by the
  recognition of specific sequences.
• RE used in molecular biology typically recognize
  (??) short (4-8bp) target sequences that are
  usually palindromic (????), and cut (??) at a
  defined sequence within those sequences.
• e.g. EcoRI

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How to name a restriction endonuclease?
EcoRI
the 1st such enzyme found
Escherichia coli Species category
R13 strain
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How to estimate the frequency of the RE in a DNA
molecule or genome?

• The random occurrence of the hexameric (?????)
  sequence
• 1/4096 (4-61/46)
• What are the frequencies if the recognition
  sequences are four (tetrameric) and eight
  (octameric) nucleotides? homework

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(The largest fragment)
(The smallest fragment)

• Consider a linear DNA molecule with 6 copies of
  GAATTC
• it will be cut into 7 fragments which
  could be separated by the gel electrophoresis.

Fig 20-3 digestionof a DNA fragment with
endonuclease EcoRI
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Use of multiple REs allows different regions of a
DNA molecule to be isolated

• A given molecule will generate a characteristic
  series of pattern when digested with a set of
  different enzymes.
• e.g. the combination of EcoRI HindIII

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(1) Restriction enzymes differ in the recognition
specificity target sites are different.(2)
Restriction enzymes differ in the length they
recognized, and thus the frequencies differ.(3)
Restriction enzymes differ in the nature of the
DNA ends they generate blunt/flush ends (???),
sticky/staggered ends (????).(4) Restriction
enzymes differ in the cleavage activity.
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Fig 20-4 Recognition sequences and cut sites of
various endonucleases
blunt ends (???)
sticky ends (????)
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Fig 20-5 Cleavage of an EcoRI site. The 5
protruding ends are said to be sticky because
they readily anneal through base-pairing to DNA
molecules cut with the same enzyme
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CHAPTER20 Techniques of Molecular Biology

• Topic 1 Nucleic acids

1. Separation by Electrophoresis (????)
2. Cut by Restriction endonuclease (????????)
3. Identification by Hybridization (????)
4. Amplification by PCR (PCR??)
5. DNA Cloning and gene expression (DNA???????)
6. Genome sequence analysis (????????)

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3. DNA hybridization can be used to identify
specific DNA molecules
Nucleic acids- DNA hybridization

• Hybridization the process of base-pairing
  between complementary ssDNA or RNA from two
  different sources.

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Probe (??)

• A labeled, defined sequence used to search
  mixtures of nucleic acids for molecules
  containing a complementary sequence.

The mixture being probed has typically either
been separated by size on a gel, or is
distributed as a library in different colonies
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A probe must be labeled before applied in
hybridization (Why?)

• It can be readily located once it has found its
  target sequence.

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Labeling (??) of DNA or RNA probes
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 put the labels at the ends Uniform
labeling put the labels internally
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End labeling
5-end labeling using polynucleotide kinase
(PNK) 3-end labeling using terminal transferase
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How to label one end of a DNA Labeling at both
ends by kinase, then remove one end by
restriction digestion.
---------------------G ---------------------CTTAAp
5
5pAATTC G
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Uniformly labeling of DNA/RNA
Nick translation labeling of DNA DNase I to
introduce random nicks into template DNA ? DNA
pol I to remove dNMPs from 3 to 5 and add new
dNMP including labeled nucleotide at the 3 ends.
Hexanucleotide primered labeling of DNA Denature
DNA ? add random hexanucleotide primers and DNA
pol ? synthesis of new strand incorporating
labeled nucleotide.
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Strand-specific RNA probes labeled by in
vitro transcription of the desired RNA sequence.
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Hybridization probes can identify
electrophoretically-separated DNAs and RNAs

• Hybridization the process of base-pairing
  between complementary ssDNA or RNA from two
  different sources.

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Electrophoresis
blotting
gel
membrane
Hybridization
Northern/Southern blot analysis
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Another illustration
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Southern and Northern blotting
DNA on blot
RNA on blot

• Genomic DNA preparation RNA
  preparation
• Restriction digestion -
• Denature with alkali -
• Agarose gel electrophoresis ?
• DNA blotting/transfer and fixation
  RNA
• 6. Probe labeling ?
• 6. Hybridization (temperature) ?
• 7. Signal detection (X-ray film or antibody)
  ?

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Northern analysis of COB RNAs in S. cerevisiae
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Comparison of Southern, Northern and Western bolt
hybridization
Blot type Target Probe Applications
Southern DNA DNA or RNA mapping genomic clonesestimating gene numbers, etc
Northern RNA DNA or RNA RNA sizes and abundance (gene expression level)
Western Protein Antibodies protein size and abundance (gene expression level)
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CHAPTER20 Techniques of Molecular Biology

• Topic 1 Nucleic acids

1. Separation by Electrophoresis (????)
2. Cut by Restriction endonuclease (????????)
3. Identification by Hybridization (????)
4. Amplification by PCR (PCR??)
5. DNA Cloning and gene expression (DNA???????)
6. Genome sequence analysis (????????)

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4. Polymerase chain reaction (PCR) amplifies DNAs
by repeated rounds of DNA replication in vitro
Nucleic acids- PCR

• PCR is to used to amplify a DNA sequence using a
  pair of primers each complementary to one end of
  the DNA target sequence.

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The PCR cycle Three different steps proceed in
each 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.

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Fig. Steps of PCR
Template
Primers
Enzymes
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The PCR amplification
Many cycles (25-35 in common) are performed to
complete one PCR reaction, which resulted in an
exponential amplification of the target DNA if
both forward and reverse primers pair.
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DNA 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. .
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PCR Primers

1. Anneal on opposite strands of the target
   sequence.
2. About 18 to 30 nt long and have similar GC
   contents so that they anneal to their
   complementary sequences at similar temperatures.
3. Tm2(at)4(gc) determine annealing
   temperature. If the primer is 18-30 nt, annealing
   temperature can be Tm ? 5oC

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5-ATTCCGATCGCTAATCGATGGC------- TCCTGTGCA
TTTCGCCACTAGAG-3 3-TAAGGCTAGCGATTAGCTACCG-------
AGGACACGTAAAGCGGTGATCTC-5
DNA sequence is written from 5 to the 3 end if
not stated. And only the sense strand is usually
given instead of both strands.
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Degenerate primers (????) an oligo pool
derived from a 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
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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. High-accuracy DNA
  polymerase is available commercially.
• To optimize PCR, the annealing temperature and
  the Mg concentration are varied, or the nested
  PCR is carried out.

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Nested PCR to increase specificity
First round primers
Gene of interest
Second round PCR
First round PCR
Second round primers
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Reverse transcriptase (RT)-PCR
5-Cap
mRNA
AAA(A)n
(dT)1218 primer
anneal
5-Cap
3
5
AAA(A)n
Reverse transcription
dNTP, RT
5-Cap
5
Regular PCR
AAA(A)n
cDNAmRNA hybrid
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PCR mutagenesis (??)
PCR can be used to introduce deletion and point
mutations

1. Two separate PCR reactions are performed.
2. One PCR amplifying the 5-portion of the insert,
   and the other amplifying the 3-portion of the
   insert.
3. The point mutation/deletion mutations are located
   in the primers

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Forward mutagenic primer
SP6 primer
T7 primer
Reverse mutagenic primer
First PCR
Remove primers Denature and anneal
3
3
Extend to full length by DNA polymerase
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Second PCR
SP6 primer
T7 primer
DNA cloning
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CHAPTER20 Techniques of Molecular Biology

• Topic 1 Nucleic acids

1. Separation by Electrophoresis (????)
2. Cut by Restriction endonuclease (????????)
3. Identification by Hybridization (????)
4. Amplification by PCR (PCR??)
5. DNA Cloning and gene expression (DNA???????)
6. Genome sequence analysis (????????)

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5. DNA cloning, analysis and gene expression
Nucleic acids- sequencing
The ability to construct recombinant DNA
molecules and maintain them in cells is called
DNA cloning.
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• Processes (??) of DNA cloning
• Form the recombinant DNA molecules (??DNA) by
  inserting your interested DNA fragments into a
  proper vector (??). (Require restriction enzymes
  and ligase)
• Transform (??) the recombinant DNA molecules into
  competent cells (?????).
• Propagation of the cells containing the
  recombinant DNA to form a clone (??), a set of
  identical cells containing the same recombinant
  DNA.
• Select the desired clones using the selective
  marker.

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1. Restriction digestion of your insert and vector
   using the same enzyme.
2. Use ligase to join your insert and vector
   together.
3. Transform the ligation products into E. coli.
   competent cells.
4. Grow the cells on a plate containing tetracycline
   (???).

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• Host organisms/cells where the plasmids get
  multiplied and propagated faithfully, which is
  crucial for DNA cloning.
• ---Prokaryotic host E. coli ( most cases)
• ---Eukaryotic host Yeast Saccharomyces
  cerevisiae (large fragments of human genome)

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• General features of a Vector
• They contain an origin of replication and can
  autonomously replicating DNA independent of
  hosts genome.
• Easily to be isolated from the host cell. Most
  are circular, some are linear (e.g. YAC vector).
• Contains at least one selective marker, which
  allows host cells containing the vector to be
  selected amongst those which do not.
• Contains a multiple cloning site (MCS) to be cut
  by restriction enzymes for DNA manipulation.

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Cloning vectors (????) allowing the exogenous
DNA to be inserted, stored, and manipulated at
DNA level. E. coli cloning vector (circular)
plasmids (??) bacteriophages (l and M13)
(???) plasmid-bacteriophage l hybrids (cosmids)
(????-?????????). Yeast cloning vector yeast
artificial chromosomes (YACs,???????) (Linear)
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• Plasmids small, extrachromosomal circular
  molecules, from 2 to 200 kb in size, which exist
  in multiple copies within the host cells.
• Contain an origin of replication, at least one
  selective marker and multiple cloning site.
• Example of selective marker ampr gene encoding
  the enzyme b-lactamse which degrades penicillin
  antibiotics such as ampicillin.
• The commonly used plasmid are small in size ( 3
  kb)

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Libraries of DNA molecules can be created by
cloning (Genomic library and cDNA library) A DNA
library (DNA??) is a population of identical
vectors that each contains a different DNA
insert. (Fig. 20-8) Genomic Library (?????) the
DNA inserts in a DNA library is derived from
restriction digestion or physical shearing of the
genomic DNA. cDNA library (cDNA??) the DNA
inserts in a DNA library is converted from the
mRNAs of a tissue, a cell type or an organism.
cDNA stands for the DNA copied from mRNA. (Fig.
20-19)
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Different Insert fragments
Fig 20-8 construction of a DNA library
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• cDNA library generation
• The mRNAs are firstly reverse transcript into
  cDNA, and these cDNA, both full length and
  partial, are cloned to make the cDNA library.

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Fig 20-9 Construction of cDNA library
anneal
Reverse transcription
Regular PCR
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Screening of positive clones
Colony screening

1. Antibiotic screening (?????) only the
   recombinant plasmids grow on the
   antibiotic-containing plate.
2. Blue-white screening (?????) DNA insertion in
   the vector shuts down the LacZ gene expression,
   and turns the colony to white.
3. Colony hybridization screening (??????) from a
   library.

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Antibiotic screening (?????) only intact
plasmids grow on the antibiotic-containing plate.
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Recombinant DNA molecules
X if the vector is in the
phosphorylated state
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Dephosphorylate the vector using alkaline
phosphate can prevent religation of vector
molecules
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Blue white screening ---Allow the discrimination
of recombinant plasmid from the religated ones
Lac promoter
Ampr
MCS (Multiple cloning sites, ?????)
pUC18 (3 kb)
lacZ
Insertion of a DNA fragment interrupts the ORF of
lacZ gene, resulting in non-functional gene
product that can not digest its substrate x-gal.
ori
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lacZ encode enzyme b-galactosidase
(substrate of the enzyme)
lac promoter?
X-gal
IPTG
Blue product
During the experiment, IPTG is added to the
selective growth medium. Insertion of the target
DNA fragment into the MCS will prevent the
formation of the blue colony, and white colony is
formed instead.
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Recreated vector blue transformants Recombinant
plasmid containing inserted DNA white
transformants
Recreated vector (no insert)
Recombinant plasmid (contain insert)
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Colony hybridization-Southern blot
Transfer to nitrocellulose or nylon membrane
Select positive from master plate
Keep master plate
Denature DNA(NaOH) Bake onto membrane
Probe with 32p-labled DNA complementary to
gene of interest
Expose to film
Screening by plaque hybridization
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Analysis of DNA clones
Analysis of a clone

1. Restriction mapping digestion of the plasmid
   prepared from a clone with restriction enzymes to
   investigate if the interested DNA is inserted the
   recombinant plasmid.
2. Sequencing the cloned DNA to see if the inserted
   DNA maintains the correct sequence.

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2 Positive clones digested with different
restriction enzymes
Empty vector
1 Kb ladder
Restriction mapping
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Sequencing
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Gene expression
Expression of a gene from a transformed/transfecte
d plasmid

1. Transformation (??) introduction of plasmids
   into bacteria.
2. Transfection (??) introduction of plasmids and
   other exogenous nucleic acids into eukaryotes
   such as mammalian cells.

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Expression vectors allowing the exogenous DNA
to be stored and expressed in an organism. --E.
coli expression vector --Yeast expression
vector --Mammalian expression vector Features In
addition to the origin of replication, selective
marker, multiple cloning site, expression vector
has to contain a promoter and terminator for
transcription. The inserted gene has to have a
start codon and a stop codon for translation
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T7 promoter
RBS
Start codon
MCS
Ampr
Transcription terminator
T7 expression vector
E. coli expression vector
ori
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H4 Eukaryotic Vectors
MCS
Insert Figure 1
Yeast expression vector
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Fusion proteins
Gene cloning and expression provides a very
powerful way to obtain a large amount of the
target protein fused with an enzyme, fluorescence
protein or a tag for identification (??) or
purification (??).
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Examples
Lac fusions (Enzyme) fuse your target gene
with the LacZ coding sequence. His-tag fusions
(Tag) A sequence encodes His-tag was inserted
at the N- or C- termini of the target ORF,
allowing the fusion protein to be purified by
binding to Ni2 column. GFP fusions
(Fluorescence protein) insert your targeted gene
at the N- or C- termini of GFP (green
fluorescence protein, ??????), and your fusion
protein will give you green fluorescence signal.
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CHAPTER20 Techniques of Molecular Biology

• Topic 1 Nucleic acids

1. Separation by Electrophoresis (????)
2. Cut by Restriction endonuclease (????????)
3. Identification by Hybridization (????)
4. Amplification by PCR (PCR??)
5. DNA Cloning and gene expression (DNA???????)
6. Genome sequence analysis (????????)

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5. Sequencing
Nucleic acids- sequencing

• 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. (Maxam and Gilbert chemical method,
  not widely used)
• 2. Chain-termination method (Sangers method,
  widely used)

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Sangers enzymic method
Maxam and Gilbert
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Chain-termination method (????)

• ddNTPs are chain-terminating nucleotides the
  synthesis of a DNA strand stops when a ddNTP is
  added to the 3 end

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The absence of 3-hydroxyl lead to the
inefficiency of the nucleophilic attack on the
next incoming substrate molecule.
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Tell from the gel the position of each G
If one ddGTP is added to 100 dGTP, DNA synthesis
aborts at a frequency of 1/100 every time the
polymerase meets a ddGTP
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Fig 20-15 DNA sequencing gel
Four separate reactions dNTP ddGTP, dNTP
ddATP dNTP ddCTP, dNTP ddTTP Each ddNTP
carries a fluorescence group, allowing us to
Read the sequence directly from the gel.
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Automatic sequencer

• Fluorescence
• Labeled ddNTP
• 2. Polymerase catalyzed

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Shotgun sequencing of a bacterial genome
(??????????)

• The bacterial genome was randomly sheared into
  many random fragments with an average size of 1
  kb, and cloned intro a vector. (Prepare what you
  are going to shot)
• Individual recombinant DNA clones are randomly
  picked to prepare DNA for sequencing on an
  automated sequencer. (shot)
• This is called shotgun sequencing.

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In use of shotgun sequencing strategy, multiple
sequence coverage is required to obtain all
genome sequence. For example The genome of
bacteria Hemophilus influenzae is 1.8 Mb, each
sequence read produces 600 bp of sequence. If
33,000 different clones were picked for
sequencing, a total of 600 bp x 33,000 20 Mb
sequence was produced.
Critical thinking how to design a pair of
primers to sequence the inserts of all the
recombinant plasmids in a genomic library?
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The shotgun strategy permits a partial assembly
of large genome sequence

• The core techniques for sequencing the large
  genomes, e.g. human, are
• automated shotgun sequencing (obtain sequence)
• then the subsequent use of computer to assemble
  the different sequences (analyze sequence, which
  is the rate-limiting step).

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Flow chart of Human genome sequencing project
2. Shotgun sequencing
1. Recombinant Plasmid Library
3. Sequence Assembly
Fig 20-16
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• Assembly Step 1 form contigs
• (A single contig is about 50,000 to 200,000 bp. )
• Sophisticated computer programs have been
  developed that assemble the short sequences from
  random shotgun DNAs into larger contiguous
  sequences called contigs.

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Assembly Step 2 The paired-end strategy permits
the assembly of larger scaffolds (1-2 Mb)
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• Fig 20-17. Contigs are linked by sequencing the
  ends of large DNA fragments (plasmid library
  containing larger DNA fragments).

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Assembly flowchart

1. Assemble the contigs from 1kb plasmid shotgun
   sequence. (50 kb-200 kb)
2. Assemble the contigs to large scaffold by
   sequencing both ends of 5 kb plasmids. (lt500 kb)
3. Assemble the larger scaffolds (gt1 Mb) by
   sequencing the end of the BAC library.

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Genome-wide analysis

• The purpose of this analysis is to predict the
  protein coding genes (???????) and other
  functional sequences (??????) in the genome.

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• For the genomes of bacteria and simple
  eukaryotes
• Finding protein coding genes Identification of
  ORF (open-reading frames).
• straightforward
• fairly effective
• but not all ORFreal protein coding genes
• key challenge is in identifying the functions of
  these genes.

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• For animal genomes with complex exon-intron
  structures, the challenge is far greater
• A variety of bioinformatics tools are required to
  identify genes and genetic composition of complex
  genomes.
• The computer programs identifying potential
  protein coding genes are based on many sequence
  criteria including the occurrence of extended
  ORFs that are flanked by appropriate 5 and 3
  splice sites.

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• Limitations of the computer methods
• one-fourths of genes cannot be identified by
  this way.
• The failure to identify promoters because the
  core promoter elements are highly degenerate
  (???). Although the transcription complex is
  smart enough to identify these elements in cell,
  we are not yet smart enough to write programs to
  identify them in silico (??,??).
• The most important method for validating
  predicted protein coding genes and identifying
  those missed by current gene finder program is
  the use of cDNA sequence data.

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• cDNA library sequencing and application
• Sequence the cDNAs prepared from a cDNA library
  using shotgun method to generate EST (expressed
  sequence tag) database.
• These ESTs are aligned onto genomic scaffolds to
  help us identify genes and to assemble larger
  scaffolds.

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Fig 20-18 Gene finder method analysis of
protein-coding regions in Ciona intestinalis (?? )
A 20-kb genome sequence (scaffold)
Predicted by a gene finder program
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• The mostly commonly used genome tool BLAST
• Finding regions of similarity between different
  protein coding genes.
• Input a query sequence (????) a stretch of amino
  acids or the DNA sequence encoding your
  interested protein function.
• Ask the computer to search for the homologous
  sequences in a protein or DNA database, and you
  will get all the available genes that may have
  the similar protein function.

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Figure 20-21 Example of the BLAST search result
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CHAPTER20 Techniques of Molecular Biology

• Topic 2 Proteins

1. Protein purification (?????)
2. Affinity chromatography can facilitate more rapid
   protein purification (??????)
3. Protein separation by PAGE gel electrophoresis
   (?????) and identification by Western analysis
4. Protein sequencing (?????)
5. Proteomics (?????)

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1. Protein purification (?????)

• The purification of individual proteins is
  critical to understanding their function.
• Although there are thousands of proteins in a
  single cell, each protein has unique properties,
  such as size, charge (??), shape, and in many
  instance, function, that make its purification
  somewhat different from others.

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• Purification of a protein requires a specific
  assay to allow you to monitor your purification
  status, which include a measure of the function
  of the protein, use of the antibody of the
  protein.

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Column chromatography is an efficient way to
purify proteins

• In this approach, protein fractions are passed
  though glass columns filled with appropriated
  modified small acrylamide or agarose beads.

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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.

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Fig 20-22-a
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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.

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Fig 20-22-b
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2. Affinity chromatography can facilitate more
rapid protein purification

• If the target protein is known to establish a
  specific and high-affinity interaction with a
  specific protein/nucleic acids/small molecule, we
  can couple this specific partner of the target
  protein to the column and thus the target protein
  will be selectively bound to the column.
• This method is called affinity chromatography.

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• Protein structure

Affinity chromatography

• Enzyme-substrate binding

• Receptor-ligand binding

• Antibody-antigen binding

• Ni2-His tag-fusion protein binding

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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.
• Disadvantage sometimes the binding is too tight
  to elute our target protein.

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• Sometimes tags (epitopes, ?????) can be added to
  the N- or C- terminal of the target protein,
  using DNA cloning method, to make the fusion
  protein.
• This allows the modified fusion proteins to be
  purified using immunoaffinity purification and a
  heterologous antibody specific for the tag.
• Importantly, the binding affinity can change
  according to the condition. e.g. the
  concentration of the Ca2 in the solution.

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Immunoprecipitation (????)

• Attach the antibody to the bead, which is then
  used to precipitate (??) a specific protein from
  a crude cell extract.
• Its a useful method to detect what proteins or
  other molecules are associated with the target
  protein.

125
3. Protein separation by PAGE gel
electrophoresis, followed by a western analysis

• 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.

126

• 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 (?????).
• Proteins from the PAGE gel can be transferred to
  a membrane, followed by a western analysis of the
  target protein by a corresponding antibody.

127
A protein gel stained by Coomassie Blue
Western analysis using two specific antibodies
--- P P P P
PTB
Beta-actin
128
4. Protein sequencing (?????)

• Two sequence method
• Edman degradation (Edman???)
• 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.

129
Edman degradation

• A chemical reaction in which the amino acids
  residues are sequentially release for the
  N-terminus of a polypeptide chain.

130

• Step 1 modify the N-terminal amino with PITC,
  which can only react with the free a-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. ??

131
Fig 20-23
132
Tandem mass spectrometry

• MS is a method in which the mass of very small
  samples of a material can be determined.

133

• Step 1 digest your target protein into short
  peptides.
• 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 5Deconvolution (??) of these data and the
  sequence will be revealed.

134
5. 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.

135
Three principle methods

• 1. 2-D gel electrophoresis for protein separation
  (?????).
• 2. MS spectrometry for the precise determination
  of the molecular weight and identify of a protein
  (?????).
• 3. Bioinformatics for assigning proteins and
  peptides to the predicted products of protein
  coding sequence in the genome (?????).

136
Fig 20-24
137
CHAPTER20 Techniques of Molecular Biology

• Topic 3 Study the interaction between protein
  and nucleic acid

1. Gel retardation assay
2. Nuclease protection assay

138
Gel retardation (????)

• A short labeled nucleic acid is mixed with a cell
  or nuclear extract expected to contain the
  binding protein. Then, samples of labeled nucleic
  acid, with and without being incubated with the
  extract, are run on a gel. The DNA-protein
  complexes are shown by the presence of slowly
  migrating bands.

139
A DNA bound with more than one protein to form a
larger complex.
DNA bound to two proteins
DNA-protein complex
Bare DNA
140
DNase I footprinting (DNase I ???)

• Identify the actual region of sequence with
  which the protein interacts.

Sequence ladder is required to determine the
precise position
AATAAG

5
141
DNase footprinting (1)The protein protects DNA
from attack by DNase. (2)Treat the DNA-protein
complex with DNase I under mild conditions, so
that an average of only one cut occur per DNA
molecule.
Bind protein
DNase(mild),then remove protein and denature DNA
Electrophoresis, autoradiograph
142
The three lanes represent DNA that was bound to
0, 1, and 5 units of protein. The lane with no
protein shows a regular ladder of fragments. The
lane with one unit shows some protection, and
the lane with 5 units shows complete protection
in the middle.By including sequencing ladders,
we can tell exactly where the protein bound.
Protein
T C G G A G C A A C G C A A A C A A A C G T G C T
T G G
1
0
5
143
CHAPTER20 Techniques of Molecular Biology

• Topic 4 Determining the Structure of
• Protein and nucleic acids

1. X-ray crystallography (X-????)
2. NMR (????)

144
X-ray crystallography and NMR Determining the
tertiary structure
X-ray crystallography

• Measuring the pattern of diffraction of a beam of
  X-rays as it pass through a crystal. The first
  hand data obtained is electron density map, the
  crystal structure is then deduced.

• A very powerful tool in understanding protein
  tertiary structure

• Many proteins have been crystallized and analyzed

145
The ribosome structure and its interaction with
mRNA and tRNA
146
NMR

• Measuring the relaxation of protons after they
  have been excited by radio frequencies in a
  strong magnetic field.

• Measure protein structure in liquid but not in
  crystal.

• Protein measured are usually smaller than 30 KDa.

147
CHAPTER20 Techniques of Molecular Biology
Nucleic acids techniques Electrophoresis
Restriction digestion Hybridization (southern
northern) PCR amplification sequencing and
genome sequencing DNA cloning and gene
expression. Protein techniques Protein
purification affinity chromatography Protein
separation and identification by western blot
Protein sequencing Proteomics. Study the
interaction between protein and nucleic acid Gel
retardation Nuclease protection
assays Determining the Structure of protein and
nucleic acids X-ray crystallography, NMR
148
CHAPTER20 Techniques of Molecular Biology
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