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Title: Welcome Each of You to My Molecular Biology Class


1
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
Part V METHODS
  • Ch 20 Techniques of
  • Molecular Biology
  • Ch 21 Model Organisms

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
CHAPTER20 Techniques of Molecular Biology
  • Topic1 Nucleic acids
  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
Electrophoresis
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
8
Electrophoresis
  • 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 supercoiledgt
    lineargt nicked or relaxed

9
Fig 20-1 DNA separation by gel electrophoresis
large
moderate
small
After electr
10
To separate DNA of different size ranges
Electrophoresis
  • Narrow size range of DNA use polyacrylamide
  • Wide size range of DNA use agarose gel
  • Very large DNA(gt30-50kb) use pulsed-field gel
    electrophoresis

11
pulsed-field gel electrophoresis
Electrophoresis
Switching between two orientations the larger
the DNA is, the longer it takes to reorient
12
Restriction endonucleases cleave DNA molecules at
particular sites
Nucleic acid
Restriction digestion
  • Why use endonucleases?
  • --To make large DNA molecules break into
    manageable fragments

13
Restriction digestion
  • 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 (????).
  • e.g. EcoRI

5.GAATTC..3 .CTTAAG.
14
Restriction digestion
  • To name a restriction endonuclease
  • e.g. EcoRI

the 1st such enzyme found
Escherichia coli Species category
R13 strain
15
Restriction digestion
  • Frequency of the occurrence of hexamaeric
    sequence
  • 1/4096 (4-6)
  • Randomly

16
(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 in the gel electrophoresis by
    size

Fig 20-3 digestionof a DNA fragment with
endonuclease EcoRI
17
Restriction digestion
  • 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

18
Different enzymes recognize their specific target
sites with different frequency
Restriction digestion
  • EcoRI Recognize hexameric sequence 4-6
  • Sau3A1 Recognize terameric sequence 4-4
  • Thus Sau3A1 cuts the same DNA molecule more
    frequently

19
Restriction digestion
blunt ends
sticky ends
Fig 20-4 recognition sequences and cut sites of
various endonucleases
20
Restriction digestion
  • 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
21
DNA hybridization can be used to identify
specific DNA molecules
Nucleic acid
DNA hybridization
  • Hybridization the process of base-pairing
    between complementary ssDNA or RNA from two
    different sources

22
  • Probe a labeled, defined sequence used to search
    mixtures of nucleic acids for molecules
    containing a complementary sequence

23
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
24
End labeling
Single stranded DNA/RNA
5-end labeling polynucleotide kinase
(PNK) 3-end labeling terminal transferase
25
(No Transcript)
26
Labeling at both ends by kinase, then remove one
end by restriction digestion
---------------------G ---------------------CTTAAp
5
5pAATTC G
27
J1 Characterization of clones
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 .
28
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
29
J1 Characterization of clones
J1-5 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)
    ?

30
Southern analysis
31
Southern bolt hybridization
32
Northern analysis COB RNAs in S. cerevisiae
33
J1 Characterization of clones
Blot type Target Probe Applications
Southern DNA DNA or RNA mapping genomic clonesestimating gene numbers
Northern RNA DNA or RNA RNA sizes, abundance,and expression
Western Protein Antibodies protein size, abundance
34
Sequencing
Nucleic acid
  • 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
Tell from the gel the position of each G
DNA synthesis aborts at a frequency of 1/100
every time the polymerase meets a ddGTP
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
NUCLEIC ACIDS
  • 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.

40
Fig 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. Its
    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 3100kb.

NUCLEIC ACIDS
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.

NUCLEIC ACIDS
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.

proteins
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, its 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.

proteins
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.

proteins
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.

proteins
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.
  • Its 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.

proteins
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-separ
ated 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

proteins
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.

proteins
65
Edman degradation
  • Its a chemical reaction in which the amino
    acids 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 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.

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 5Deconvolution 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.

proteins
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.

NUCLEIC ACIDS
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,
    3000040000 clones are needed, which is ten times
    larger as the genome. This is called 10sequence
    coverage.
  • This method might seem tedious, but its much
    faster and cheaper than the digestion-mapping-sequ
    encing method. As the computer is much faster at
    assembling sequence than the time required to map
    the chromosome.

76
J3 Polymerase chain reaction
Analysis and uses of cloned DNA
  • J3-1 PCR
  • J3-2 The PCR cycle
  • J3-3 Template
  • J3-4 Primers
  • J3-5 Enzymes
  • J3-6 PCR optimization


77
J3-1 PCR
J3 Polymerase chain reaction
  • 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.

78
J3-2 The PCR cycle
J3 Polymerase chain reaction
  • 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.

79
J3 Polymerase chain reaction
80
J2 nucleic acid sequencing
Fig. Steps of PCR
Template
Primers
Enzymes
81
J3 Polymerase chain reaction
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.

82
J3-4 Primers
J3 Polymerase chain reaction
  • PCR primers need to be about 18 to 30 nt long and
    have similar GC contents so that they anneal to
    their complementary sequences at similar
    temperatures.They are designed to anneal on
    opposite strands of the target sequence.
  • Tm2(at)4(gc) determine annealing
    temperature. If the primer is 18-30 nt, annealing
    temperature can be Tm?5oC

83
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
84
J3-56 Enzymes and PCR Optimization
J3 Polymerase chain reaction
  • 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.

85
J2 nucleic acid sequencing
PCR optimization
I.Reverse transcriptase-PCR
II.Nested PCR
86
Fig Nested PCR
J2 nucleic acid sequencing
First round primers
Gene of interest
Second round PCR
First round PCR
Second round primers
87
J2 nucleic acid sequencing
Reverse transcriptase-PCR
Fig RT-PCR
5-Cap
mRNA
AAA(A)n
(dT)1218 primer
anneal
5-Cap
3
5
AAA(A)n
Reverse transcriptase
dNTP
5-Cap
5
Regular PCR
AAA(A)n
cDNAmRNA hybrid
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