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