Techniques of molecular biology

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Chapter 20
techniques of molecular biology

O4级生物学基地班
林青青200431060021
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Introduction
The methods depend upon, and were developed from, an understanding of the properties of biological macromolecules themselves.

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Topic 1
nucleic acids
Electrophoresis
Restriction
Hybridization
DNA Cloning and gene expression
PCR
Genome sequence & analysis
Comparative genome analysis
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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
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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
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Fig 20-1: DNA separation by gel electrophoresis
http://a32.lehman.cuny.edu/molbio_course/agarose.htm
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Some fundamental steps of electrophoresis
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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
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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: supercoiled> linear> nicked or relaxed
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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
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To name a restriction endonuclease
e.g. EcoRI
Escherichia coli
Species category
R13
strain
the 1st such
enzyme found
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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
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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
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Recognition sequences and cut sites of various endonucleases
blunt ends
sticky ends
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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
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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
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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 αorγ32P
nonradioactive biotin, digoxigenin, fluorescent dye
In a single stranded form for hybridization

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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
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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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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
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Ways of Molecular Hybridization
A. Transfer blotting (转移印迹)
Southern blotting
Northern blotting
Western blotting
Eastern blotting

B. Dot blotting & Slot blotting (点印迹, 狭缝印迹)

C. In situ hybridization (原位杂交)
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Southern and Northern blotting

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) 
DNA on blot RNA on blot
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Characteristics of transfer bloting
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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

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Southern analysis
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DNA 样品



DNA 探针
变性
X-ray 片
限制性内切酶消化
琼脂糖凝胶电泳
转移印迹
胶 膜
两部分工作
标记
杂交
暴光
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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
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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
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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
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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
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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
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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
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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
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DNA library
A population of identical vectors that each contains a different DNA insert

Genomic library (the simplest)
cDNA library
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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: Ta=Tm-5 C



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(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.
J3 Polymerase chain reaction
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(2) Primers
PCR primers need to be about 18 to 30 nt long and have similar G+C contents so that they anneal to their complementary sequences at similar temperatures.They are designed to anneal on opposite strands of the target sequence.
Tm=2(a+t)+4(g+c): determine annealing temperature. If the primer is 18-30 nt, annealing temperature can be Tm5oC
J3 Polymerase chain reaction
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(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
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(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.
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Rate of PCR 2n
Initial
DNA

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4
2
1
Number of DNA molecules
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PCR optimization
I.Reverse transcriptase-PCR
II.Nested PCR
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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
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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

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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
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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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DNA synthesis aborts at a frequency of 1/100 every time the polymerase meets a ddGTP
Tell from the gel the position of each G
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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.
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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, 30000~40000 clones are needed, which is ten times larger as the genome. This is called 10×sequence coverage.
This method might seem tedious, but it’s much faster and cheaper than the digestion-mapping-sequencing method. As the computer is much faster at assembling sequence than the time required to map the chromosome.
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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
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Fig 20-16
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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 3~100kb.
The end of each clone can be sequenced easily, and these larger clones can firstly assemble together.
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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
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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.
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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
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Topic 2
proteins
Purification
Separation
Sequencing
proteomics
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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
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(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
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(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
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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
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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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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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(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.
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Affinity
chromatography
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Immunoaffinity chromatography
An antibody that is specific for the target is attached to the bead, and ideally only the target protein can bind to the column.
However, sometimes the binding is too tight to elute our target protein, unless it is denatured. But the denatured protein is useless.
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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.
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immunoprecipitation
We attach the antibody to the bead, and use it to precipitate a specific protein from a crude cell extract.
It’s a useful method to detect what proteins or other molecules are associated with the target protein.
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(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
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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
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(4) Antibodies visualize electrophoretically-separated 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
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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).
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Edman degradation
It’s a chemical reaction in which the amino acid’s residues are sequentially release for the N-terminus of a polypeptide chain
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Step 1: modify the N-terminal amino with PITC, which can only react with the free α-amino group.
Step 2: cleave off the N-terminal by acid treatment, but the rest of the polypeptide remains intact.
Step 3: identify the released amino acids by High Performance Liquid Chromatography (HPLC).
The whole process can be carried out in an automatic protein sequencer.
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Tandem mass spectrometry
MS is a method in which the mass of very small samples of a material can be determined.
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Step 1: digest your target protein into short peptide.
Step 2: subject the mixture of the peptide to MS, and each individual peptide will be separate.
Step 3: capture the individual peptide and fragmented into all the component peptide.
Step 4: determine the mass of each component peptide.
Step 5: Deconvolution of these data and the sequence will be revealed.
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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
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Proteomics is based on three principal methods:
two-dimensional gel electrophoresis for protein separation
Mass spectrometry for the precise determination of the molecular weight and identity of a protein
Bioinformatics for assigning proteins and peptides to the predicted products of protein-coding sequences in the genome
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The End