Chapter 20 techniques of molecular biology

1
Chapter 20 techniques of molecular biology

• O4???????
• ???200431060021

2
Introduction

• The methods depend upon, and were developed from,
  an understanding of the properties of biological
  macromolecules themselves.

3
(No Transcript)
4
Topic 1 nucleic acids

• Electrophoresis
• Restriction
• Hybridization
• DNA Cloning and gene expression
• PCR
• Genome sequence analysis
• Comparative genome analysis

5
1.Electrophoresis through a Gel separate DNA and
RNA molecules according to size

• Gel matrix
• an inert, jolly-like porous material that
  sieve the DNA molecules according to its volumn
• DNA characteristics
• negatively charged, when subject to an
  electrical field, it migrates through the gel
  toward the positive pole

6
Two types of normal gel matrices

• Polyacrylamide
• has high resolving capability but can separate
  DNAs only over a narrow size range
• Agarose
• has less resolving power than polyacrylamide
  but can separate from one another DNA molecules
  of up to tens, and even hundreds, of kilobases

7
Fig 20-1 DNA separation by gel electrophoresis
http//a32.lehman.cuny.edu/molbio_course/agarose.h
tm
8
Some fundamental steps of electrophoresis
9
Whereas very long DNAs are unable to penetrate
the pores in agarose

• DNA molecules above a certain size (30 to 50 kb)
  usually use pulsed-field electrophoresis to
  separate

10
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

11
2.Restriction endonucleases cleave DNA molecules
at particular sites

• endonucleases
• --To make large DNA molecules break into
  manageable fragments
• 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

12
To name a restriction endonuclease

• e.g. EcoRI

Escherichia coli Species category
R13 strain
the 1st such enzyme found
13

• 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

14
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

15
Recognition sequences and cut sites of various
endonucleases
blunt ends
sticky ends
16
(No Transcript)
17
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

18
3.DNA hydridization can be used to identify
specific DNA molecules

• Hybridization
• the process of base-pairing between
  complementary single-stranded polynucleotides
  from two different sources

19
probe

• Notes
• Probe is a specific DNA or RNA fragment which
  can bind with the sample DNA or RNA for
  detection. ATCCGATCG--------
• Source of probe
• synthesized, cloning genomic DNA or cDNA, as
  well as RNA.
• Probe must be labeled before hybridization.
• radioactive aor?32P
• nonradioactive biotin, digoxigenin,
  fluorescent dye
• In a single stranded form for hybridization

20
There are two basic mothods for labeling DNA

• Synthesizing new DNA in the presence of a labeled
  precursor

• Adding a label to the end of an intact DNA
  molecule

21
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

22
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

23
Ways of Molecular Hybridization

• A. Transfer blotting (????)
• Southern blotting
• Northern blotting
• Western blotting
• Eastern blotting
• B. Dot blotting Slot blotting (???, ????)
• C. In situ hybridization (????)

24
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 ?
• 7. Hybridization (temperature) ?
• 8. Signal detection (X-ray film or antibody)
  ?

25
Characteristics of transfer bloting
Blot type Target Probe Applications
Southern DNA DNA or RNA mapping genomic clonesestimating gene numbers
Northern RNA DNA or RNA RNA sizes, expression abundance,and
Western Protein Antibodies protein size, abundance
26
Southern blotting

• It is first proposed by Dr. Edwin Southern in
  Edinburgh University in 1975, and term Southern
  blotting is named for him.
• Major steps electrophoresis
• transfer blotting
• molecular hybridization

27
Southern analysis
28
?????
DNA ??
DNA ??
??
????????

??
???????
??
???? ? ?
X-ray ?
??
29
Northern blot hybriodization

• Can be used to identify a particular mRNAs
• The protocol is fairly similar to that describe
  for southern blotting except that mRNA are not
  needed to be digested with any enzymes
• An experimenter might carry out northern blot
  hybridization to ascertain the amount of a
  particular mRNA present in a sample rather than
  its size
• Moreover, northern blot hybridization might be
  carried out to compare the relative levels of a
  particular transcript between tissues of an
  organism

30
4. DNA cloning

• DNA cloning
• the ability to construct recombinant DNA
  molecules and maintain them in cells
• This process typically involves a vector that
  provides the information necessary to propagate
  the cloned DNA in the cell and an insert DNA that
  is inserted within the vector and includes the
  DNA of interest

31
5. PCR

• The polymerase chain reaction (PCR) amplifies
  DNAs by repeated rounds of DNA replication in
  vitro
• PCR
• is used to amplify a sequence of DNA using a
  pair of primers each complementary to one end of
  the DNA target sequence

32
Cloning DNA in plasmid vectors

• Vector DNAs typically have three characteristics
• An origin of replication that allow them to
  replicate independently of the chromosome of the
  host
• A selectable marker that allows cells that
  contain the vector to be readily identified
• Single sites for one or more restriction enzymes
  that allow DNA fragments to be inserted at a
  defined point within an otherwise intact vector

33
Vector DNA can be introduced into host organisms
by transformation

• Transformation
• the process by which a host organism can take
  up DNA from its environment

34

• Genetic competence
• An antibiotic to which the plasmid imparts
  resistance is then used to select transformants
  that have acquired the plasmid
• Transformation generally is a relatively
  inefficient process

35
Libraries of DNA molecules can be created by
cloning

• Generate a specific clone
• If the starting donor DNA is simple
• ----restriction enzyme gel electrophoresis
• If the starting DNA is more complex
• ----clone the whole population of fragment
  separate the individual clones

36
DNA library

• A population of identical vectors that each
  contains a different DNA insert
• Genomic library (the simplest)
• cDNA library

37
Polymerase Chain Reaction

• The PCR consists of three defined sets of
  temperatures and times termed steps
• (1) denaturing, (2) annealing, (3) extension.
• Denaturing 940C 45 Sec
• Annealing 550C-630C 30 Sec 30
  cycles
• Extension 720C 45 Sec
• Annealing temperature TaTm-5 ?C

38
J3 Polymerase chain reaction
(1) 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.

39
(2) 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

40
(3) A pair of primers

• The key to the PCR lies in the design of
  the primers
• A.20-30 bp in length with each
  complementary
• to the 3 side in a strand of target
  DNA.
• B. not self-complementary
• C. not consecutive 4 same bases (AAAA)
• D. proper GC content (40-60)
• primer sequence from Genbank,
• designed
  by software

41
(4) DNA polymerases (Taq polymerases)

• It is thermostable, temperature optimum
• is 720C and active when the temperature
• over 960C.
• It was first isolated from the thermophilic
  bacterium (Thermus aquaticus )found in hot
  springs.

42
(No Transcript)
43

• Rate of PCR 2n

InitialDNA
8
4
2
1
Number of DNA molecules
44
PCR optimization

• I.Reverse transcriptase-PCR

II.Nested PCR
45
Similarity and difference between DNA cloning and
PCR

• Similarity
• repeated rounds of DNA duplication

• Difference
• DNA cloning --- rely on a selective reagent
  or other device to locate the amplified sequence
  in an already existing library of clones
• PCR --- the selective reagent, the pair of
  oligonucleotides, limits the amplification
  process to the particular DNA sequence of
  interest from the beginning

46
5.Genome sequence analysis

• Nested sets of DNA fragments reveal nucleotide
  sequences
• Shotgun sequencing a bacterial genome
• The shotgun strategy permits a partial assembly
  of large genome sequences
• The paired-end strategy permits the assembly of
  large genome scaffolds

47
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.
• 2. chain-termination method

48
chain-termination method

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

49
The absence of 3-hydroxyl lead to the
inefficiency of the nucleophilic attack on the
next incoming substrate molecule
50
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
51
Shotgun sequencing a bacterial genome

• The bacterium Hemophilus influenzae was the first
  free-living organism to have a complete genome
  sequence and assembly
• This organism is chosen as its genome is small
  (1.8Mb) and compact
• Its whole genome was sheared into many random
  fragments with an average length of 1kb.

52

• 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.
• 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.

53
The shotgun strategy permits a partial assembly
of large genome sequence

• Recombinant DNA can be rapidly isolated from
  bacterial plasmids and then quickly using the
  automated sequencing machines
• Sophisticated computer programs have been
  developed that assemble the short sequence from
  random shotgun DNAs into large contiguous
  sequence called contigs

54
Fig 20-16
55
The paired-end strategy permits the assembly of
large genome scaffolds

• The main limitation to producing large contigs is
  the occurrence of repetitive sequence.
• 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.
• The end of each clone can be sequenced easily,
  and these larger clones can firstly assemble
  together.

56
Genome-wide analyses

• The purpose of this analysis is to predict the
  coding sequence and other functional sequence in
  the genome
• For animal genomes, a variety of bioinformatics
  tools are required to identify genes and other
  functional fragments. But the accuracy is low

57

• 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.

58
6. Comparative genome analysis

• The comparison of different animal genomes
  permits a direct assessment of changes in gene
  structure and sequence that have arisen during
  evolution
• One of the striking findings of comparative
  genome analysis is the high degree of synteny,
  conservation in genetic linkage, between
  distantly related animals.
• The most commonly used genome tool is BLAST

59
Topic 2proteins

• Purification
• Separation
• Sequencing
• proteomics

60
1. purification

• (1) specific proteins can be purified from cell
  extracts

• The purification of individual proteins is
  critical to understanding their function
• Each protein has unique properties that make its
  purification somewhat different
• The purification of a protein is designed to
  exploit its unique characteristics, including
  size, charge, and in many instances, function

61
(2) Purification of a protein require a specific
assay

• To purify a protein requires that you have an
  assay that is unique to that protein
• In many instance, it is more convenient to use a
  more direct measure for the function of the
  protein
• Incorporation assay
• are useful for monitoring the purification and
  function of many different enzymes

62
(3) Preparation of cell extracts containing
active proteins

• Most extract preparation and protein purification
  is performed at 4?C
• Cell extracts are prepared in a number of
  different ways
• Exa cells can be lysed by detergent, shearing
  forces, treatment with low ionic salt, or rapid
  changes in pressure

63
2.Separation

• (1) proteins can be separated from one another
  using column chromatography

• Column chromatography
• in this approach to protein purification,
  protein fractions are passed through glass column
  filled with appropriately modified small
  acrylamide or agarose beads.
• There are various ways columns can be used to
  separate proteins

64
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.

65
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.

66
(2) 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.

67
Affinity chromatography
68
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.

69

• 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.

70
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.

71
(3) Separation of proteins on polyacrylamide

• Proteins have neither a uniform negative nor a
  uniform secondary structure
• if, however, a protein is treated with the strong
  ionic detergent sodium dodecyl sulphate (SDS) and
  a reducing agent, such as mercaptoethanol, the
  secondary, tertiary, and quarternary structure is
  usually eliminated

72
SDS ions coat the polypeptide chain and thereby
impart on it a uniform negative charge
Mercaptoethanol reduces disulphide bonds and
thereby disrupts intramolecular and
intramolecular disulphide bridges formed between
cysteine residues
Thus, as is the case with mixtures of DNA and
RNA, electrophoresis in the presence of SDS can
be used to resolve mixtures of proteins according
to the length of individual polypeptide chains
After electrophoresis, the proteins can be
visualized with a stain,such as Coomassie
brilliant blue, that binds to protein
73
(No Transcript)
74
(4) Antibodies visualize electrophoretically-separ
ated protein

• 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

75
3. sequencing

• Protein molecules can be directly sequenced

• 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.

Two sequence method Edman degradation Tandem
mass spectrometry(MS/MS).
76
Edman degradation

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

77

• 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.

78
Tandem mass spectrometry

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

79

• 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.

80
4. proteomics

• Proteomics is concerted with the identification
  of the full set of proteins produced by a cell or
  tissue under a particular set of conditions,
  their relative abundance, and their interacting
  partner proteins

81
Proteomics is based on three principal methods

1. two-dimensional gel electrophoresis for protein
   separation
2. Mass spectrometry for the precise determination
   of the molecular weight and identity of a protein
3. Bioinformatics for assigning proteins and
   peptides to the predicted products of
   protein-coding sequences in the genome

82
The End