Molecular Biology. Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods. Part V: METHODS. Ch 20: Techniques of Molecular Biology Ch 21: Model Organisms. Molecular Biology Course.
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Part I: Chemistry and Genetics
Part II: Maintenance of the Genome
Part III: Expression of the Genome
Part IV: Regulation
Part V: Methods
Ch 20: Techniques of
Ch 21: Model Organisms
Preparation, analysis and manipulation of nucleic acids and proteins
The methods depend upon, and were developed from, an understanding of the properties of biological macromolecules themselves.
CHAPTER20: Techniques of Molecular Biology understanding of the properties of biological macromolecules themselves.
Topic 1: Nucleic acids
Gel Electrophoresis （ understanding of the properties of biological macromolecules themselves.凝胶电泳）1. Gel electrophoresis separates DNA and RNA molecules according to size, shape and topological properties
DNA gel mobility understanding of the properties of biological macromolecules themselves.(DNA在胶上的迁移性)
1.DNA and RNA molecules are negatively charged, thus move in the gel matrix (胶支持物) toward the positive pole (正电极).
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 (超螺旋)> linear (线性) > nicked or relaxed (缺刻或松散)
DNA can be visualized by staining the gel with fluorescent dyes, such as ethidium bromide (EB 溴化乙锭)Fig 20-1: DNA is separatedby gel electrophoresis
Gel matrix ( dyes, such as ethidium bromide (EB 胶支持物)
Gel matrix (胶支持物) is an inserted, jello-like porous material that supports and allows macromolecules to move through.
Polyacrylamide dyes, such as ethidium bromide (EB (聚丙稀酰胺):
has high resolving capability, and can resolve DNA/RNA that differ from each other as little as a single base pair/nucleotide.
but can separate DNA over a narrow size range (up to a few hundred bp/nt).
4 dyes, such as ethidium bromide (EB kb
a much less resolving power than polyacrylamide,
but can separate DNA molecules of up to tens of kb
Pulsed-field gel electrophoresis dyes, such as ethidium bromide (EB (脉冲电泳)
The electric field is applied in pulses that are oriented orthogonally (直角地) to each other.
Separate DNA molecules according to their molecule weight, as well as to their shape and topological properties.
Can effectively separate DNA molecules over 30-50 kb and up to several Mb in length.
Switching between two orientations: the larger the DNA is, the longer it takes to reorient
Electrophoresis is also used to separate RNAs dyes, such as ethidium bromide (EB
RNA have a uniform negative charge as DNA does.
RNA is single-stranded and have extensive secondary and tertiary structure, which significantly influences their electrophoretic mobility.
RNA can be treated with reagent such as glyoxal (乙二醛) to prevent RNA base pairing, so that its mobility correlates with the molecular weight
Nucleic acids-Restriction digestion dyes, such as ethidium bromide (EB2. Restriction endonucleases (限制性内切酶)cleave DNA molecules at particular sites
--To make large DNA molecules break into manageable fragments.
5 dyes, such as ethidium bromide (EB ’….GAATTC.….3’
How to name a restriction endonuclease? dyes, such as ethidium bromide (EB
the 1st such
The random occurrence of the hexameric (六核苷酸的)sequence:
What are the frequencies if the recognition sequences are four (tetrameric) and eight (octameric) nucleotides? [homework]
(The largest fragment) genome?
(The smallest fragment)
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
Use of multiple REs allows different regions of a DNA molecule to be isolated
(1) Restriction enzymes differ in the molecule to be isolatedrecognition 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 (平末端), sticky/staggered ends (粘性末端).(4) Restriction enzymes differ in the cleavage activity.
Fig 20-4 Recognition sequences and cut sites of various endonucleases
Fig 20-5 Cleavage of an endonucleasesEcoRI 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
Nucleic acids- DNA hybridization endonucleases3. DNA hybridization can be used to identify specific DNA molecules
Hybridization: the process of base-pairing between complementary ssDNA or RNA from two different sources.
Probe ( endonucleases探针)
A labeled, defined sequence used to search mixtures of nucleic acids for molecules containing a complementary sequence.
Labeling ( endonucleases标记) of DNA or RNA probes (why labeling?)
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
End labeling endonucleases
5’-end labeling usingpolynucleotide kinase (PNK)
3’-end labeling using terminal transferase
How to label one end of a DNA: endonucleasesLabeling at both ends by kinase,then remove one end by restriction digestion
Uniformly labeling of DNA/RNA endonucleases
Nick translation labeling of DNA:
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 of DNA:Denature DNA add random hexanucleotide primers and DNA pol synthesis of new strand incorporating labeled nucleotide.
Strand-specific RNA probes: endonucleases
labeled by in vitro transcription of the desired RNA sequence.
Southern and Northern blotting endonucleases
DNA on blot
RNA on blot
Southern analysis endonucleases
Northern analysis COB RNAs in S. cerevisiae
Comparison of Southern, Northern and Western bolt hybridization
Nucleic acids- PCR hybridization4. 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.
The PCR cycle: hybridization
Three different steps proceed in each PCR cycle.
Steps of PCR
The PCR amplification hybridization
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.
DNA template hybridization
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. .
PCR Primers hybridization
5’-ATTCCGATCGCTAATCGATGGC------- TCCTGTGCA TTTCGCCACTAGAG-3’
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.
Degenerate primers ( hybridization简并引物):
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
N= any base
Enzymes hybridization and PCR Optimization
Nested PCR hybridization
Gene of interest
Reverse transcriptase (RT)-PCR hybridization
PCR mutagenesis ( hybridization诱变)
To introduce deletion or point mutations
Forward mutagenic primer hybridization
Reverse mutagenic primer
Denature and anneal
Extend to full length by DNA polymerase
Second PCR hybridization
Nucleic acids- sequencing hybridization5. Sequencing
Two ways for sequencing:
Sanger’s enzymic method hybridization
Maxam and Gilbert
The absence of 3’-hydroxyl lead to the inefficiency of the nucleophilic attack on the next incoming substrate molecule.
Tell from the gel the position of each G the nucleophilic attack on the next incoming substrate molecule.
If one ddGTP is added to 100 dGTP, DNA synthesis aborts at a frequency of 1/100 every time the polymerase meets a ddGTP
Four separate reactions
Each ddNTP carries a fluorescence group, allowing us to “Read” the sequence directly from the gel.
Automatic the nucleophilic attack on the next incoming substrate molecule.
10x Coverage example: the nucleophilic attack on the next incoming substrate molecule.
If the H. influenzae genome is 1.8 kb, each read produces 600 bp of sequence, and 600 bp x 33,000 different colonies= 20 Mb.
That is to say 33,000 colonies are picked to prepare plasmid for sequencing.
1. Recombinant the nucleophilic attack on the next incoming substrate molecule.
2. Shotgun sequencing
3. Sequence Assembly
Assembly Step 1: form contigs the nucleophilic attack on the next incoming substrate molecule.
(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.
Fig 20-17. Contigs are linked by sequencing the ends of the nucleophilic attack on the next incoming substrate molecule.large DNA fragments (plasmid library containing larger DNA fragments).
Assembly flowchart the nucleophilic attack on the next incoming substrate molecule.
The purpose of this analysis is to predict the protein coding genes (蛋白质编码基因) and other functional sequences (其他功能序列) in the genome.
For the genomes of the nucleophilic attack on the next incoming substrate molecule.bacteria and simple eukaryotes：
Finding protein coding genes = Identification of ORF (open-reading frames).
but not all ORF=real protein coding genes;
key change is in identifying the functions of these genes
For the nucleophilic attack on the next incoming substrate molecule.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.
Limitations of the computer methods: the nucleophilic attack on the next incoming substrate molecule.
~ one-fourths of genes cannot be identified by this way.
The failure to identify promoters because the core promoter elements are highly degenerate (退变的). 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 (硅片，人工).
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.
cDNA library generation, sequencing and application: the nucleophilic attack on the next incoming substrate molecule.
Fig 20-9 RT-PCR the nucleophilic attack on the next incoming substrate molecule.
Fig 20-18 Gene finder method: analysis of protein-coding regions in Ciona intestinalis (海鞘 )
A 20-kb genome sequence (scaffold)
Predicted by a gene finder program
The comparison of different animal genomes：
One of the striking findings of comparative genome analysis is the high degree of synteny (conservation in genetic linkage,遗传连锁的保守性) between distantly related animals.
Other functional sequences under variation constraints ( is the 变异限制)：
The logic is that “functional sequence cannot be changed randomly”.
Regulatory sequences-transcription factor binding sites and larger elements of gene regulation, such as enhancers.
The computer program VISTA aligns the sequence contained in different genomes over short windows (10-20 bp), and can be used to predict the conserved regulatory sequence.
It is predicted that human and mice contain more like 50,000-100,000 enhancers.
An example of using different programs to predict the conserved regulatory sequences
The mostly commonly used genome tool conserved regulatory sequences BLAST ：
Finding regions of similarity between different protein coding genes.
Input a query sequence (询问序列): a stretch of amino acids or the DNA sequence encoding your interested protein function.
Ask the computer to search the homologous sequences in the database, and you will get all the available genes that may have the similar protein function.
Figure 20-21 Example of the BLAST search result conserved regulatory sequences
Nucleic acids- sequencing conserved regulatory sequences6. DNA cloning, analysis and gene expression
The ability to construct recombinant DNA molecules and maintain them in cells is called DNA cloning.
Cloning vectors ( same enzyme.克隆载体):allowing the exogenous DNA to be inserted, stored, and manipulated at DNA level.
E. coli cloning vector (circular):
bacteriophages (l and M13) (噬菌体)
plasmid-bacteriophage l hybrids (cosmids) (考斯质粒,质粒和噬菌体杂和体).
Yeast cloning vector: yeast artificial chromosomes (YACs，酵母人工染色体) (Linear)
Libraries of DNA molecules can be created by cloning same enzyme.
(Genomic library and cDNA library)
A DNA library (DNA文库) is a population of identical vectors that each contains a different DNA insert. (Fig. 20-8)
Genomic Library (基因组文库) : the DNA inserts in a DNA library is derived from restriction digestion or physical shearing of the genomic DNA.
cDNA library (cDNA文库) : 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)
Different Insert fragments same enzyme.
Screening of positive clones same enzyme.
Antibiotic screening ( same enzyme.抗生素选择)： only the recombinant plasmids grow on the antibiotic-containing plate.
Recombinant DNA molecules same enzyme.
if the vector is phosphorylated
Dephosphorylate the vector using alkaline phosphate to prevent religation of vector molecules
Blue white screening prevent religation of vector molecules
MCS (Multiple cloning sites,
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.
lacZ prevent religation of vector molecules encode enzymeb-galactosidase
(substrate of the enzyme)
The expression of active b-galactosidase has to be vector dependent for the selection purpose
lacZ’: a shortened derivative of lacZ,
encoding N-terminal a-peptide of b-galactosidase.
Host strain for vectors containing lacZ’:
contains a mutant gene encoding only the C-terminal portion of b-galactosidase which can then complement the a-peptide to produce the active enzyme
Recreated vector prevent religation of vector molecules: blue transformants
Recombinant plasmid: containing inserted DNA: white transformants
Recreated vector (no insert)
Recombinant plasmid (contain insert)
Colony hybridization-Southern blot prevent religation of vector molecules
Transfer to nitrocellulose
or nylon membrane
from master plate
Bake onto membrane
Probe with 32p-labled DNA
gene of interest
Expose to film
Screening by plaque hybridization
Analysis of DNA clones prevent religation of vector molecules
Analysis of a clone
1 Kb+ ladder
Gene expression enzymes
Expression of a gene from a transformed/transfected plasmid
Expression vectors: enzymes
allowing the exogenous DNA to be stored and expressed in an organism.
--E. coli expression vector
--Yeast expression vector
--Mammalian expression vector
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
T7 promoter enzymes
T7 expression vector
H4 Eukaryotic Vectors enzymes
A YEp vector
Insert Figure 1
Fusion proteins enzymes
Lac fusions: fuse your target gene with the LacZ coding sequence
His-tag fusions: A sequence encodes His-tag was inserted at the N- or C- termini of the target ORF, which allows purification of the fusion protein to be purified by binding to Ni2+ column.
GFP fusions: insert your targeted gene at the N- or C- termini of GFP, and your fusion protein will give you green fluorescence signal.
Topic 2: Proteins
1. Protein purification ( enzymes蛋白质纯化)
In this approach, protein fractions are passed though glass columns filled with appropriated modified small acrylamide or agarose beads.
Fig 20-22-a enzymes
Fig 20-22-b enzymes
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.
A protein gel stained by Coomassie Blue by a western analysis
Western analysis using two specific antibodies
--- P+ P++ P+++ P++++
Tandem mass spectrometry (MS/MS) (串连质谱).
A chemical reaction in which the amino acid’s residues are sequentially release for the N-terminus of a polypeptide chain.
The whole process can be carried out in an automatic protein sequencer. 特贵
Fig 20-23 only react with the free α-amino group.
1. 2-D gel electrophoresis for protein separation (蛋白质分离).
2. MS spectrometry for the precise determination of the molecular weight and identify of a protein (蛋白质鉴定).
3. Bioinformatics for assigning proteins and peptides to the predicted products of protein coding sequence in the genome (蛋白质确定).
Fig 20-24 only react with the free α-amino group.
CHAPTER20: Techniques of Molecular Biology only react with the free α-amino group.
Topic 3: Study the interaction between protein and nucleic acid
DNA bound to
Identify the actual region of sequence with which the protein interacts.
Sequence ladder is required to determine the precise position
DNase footprinting complex.（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.
protein and denature DNA
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.
CHAPTER20: Techniques of Molecular Biology units of protein.
Topic 4: Determining the Structure of
Protein and nucleic acids
X-ray crystallography units of protein. and NMR
Determining the tertiary structure
Nucleic acids techniques:
Electrophoresis; Restriction digestion; Hybridization (southern & northern); PCR amplification; sequencing and genome sequencing; DNA cloning and gene expression.
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