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
Part V: METHODS Ch 20: Techniques of Molecular Biology Ch 21: Model Organisms
Molecular Biology Course Chapter 20 Techniques of Molecular Biology 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. • Hybridization---the base-pairing characteristics of DNA and RNA • DNA cloning--- DNA polymerase, restriction endonucleases and DNA ligase • PCR---Thermophilic DNA polymerase
CHAPTER20: Techniques of Molecular Biology Topic 1: Nucleic acids • Separation by Electrophoresis (电泳分离) • Cut by Restriction endonuclease (限制性内切酶切割) • Identification by Hybridization (杂交鉴定) • PCR • Genome sequence & analysis • DNA Cloning and gene expression
Gel Electrophoresis （凝胶电泳） 1. Gel electrophoresis separates DNA and RNA molecules according to size, shape and topological properties
DNA gel mobility (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 moderate large small
Gel matrix (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(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 kb 3 kb 2 kb 1 kb 0.5 kb Agarose (琼脂糖): a much less resolving power than polyacrylamide, but can separate DNA molecules of up to tens of kb
Pulsed-field gel electrophoresis (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.
Fig. 20-2 pulsed-field gel electrophoresis Switching between two orientations: the larger the DNA is, the longer it takes to reorient
Electrophoresis is also used to separate RNAs 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 2. Restriction endonucleases (限制性内切酶)cleave DNA molecules at particular sites • Why use endonucleases? --To make large DNA molecules break into manageable fragments.
5’….GAATTC.….3’ ….CTTAAG…. • Restriction endonucleases (RE) arethe nucleases that cleave DNA atparticular sites by the recognitionof specific sequences. • RE used in molecular biology typically recognize (识别)short (4-8bp) target sequences that are usually palindromic (回文结构), and cut (切割) at a defined sequence within those sequences. e.g. EcoRI
How to name a restriction endonuclease? EcoRI the 1st such enzyme found Escherichia coli Species category R13 strain
How to estimate the frequency of the RE in a DNA molecule or genome? The random occurrence of the hexameric (六核苷酸的)sequence: 1/4096 (4-6) What are the frequencies if the recognition sequences are four (tetrameric) and eight (octameric) nucleotides? [homework]
(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 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 • A given molecule will generate a characteristic series of pattern when digested with a set of different enzymes. • e.g. the combination of EcoRI + HindIII
(1) Restriction enzymes differ in the recognition 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 blunt ends (平末端) sticky ends (粘性末端)
Fig 20-5 Cleavage of an EcoRI 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 3. 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 (探针) A labeled, defined sequence used to search mixtures of nucleic acids for molecules containing a complementary sequence.
Labeling (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 5’-end labeling usingpolynucleotide kinase (PNK) 3’-end labeling using terminal transferase
How to label one end of a DNA: Labeling at both ends by kinase,then remove one end by restriction digestion ---------------------G ---------------------CTTAAp5’ 5’pAATTC G
Uniformly labeling of DNA/RNA 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: labeled by in vitro transcription of the desired RNA sequence.
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)
bI1 bI2 bI3 bI4 bI5 Pre-mRNAs mRNA Northern analysis COB RNAs in S. cerevisiae
Comparison of Southern, Northern and Western bolt hybridization
Nucleic acids- PCR 4. 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: Three different steps proceed in each PCR cycle. • Denaturation (变性):The target DNA (template) is separated into two stands by heating to 95℃ • Primer annealing (退火):The temperature is reduced to around 55℃ to allow the primers to anneal. • Polymerization (elongation, extension) (延伸):The temperature is increased to 72℃ for optimal polymerization step which uses up dNTPs and required Mg++.
Fig. Steps of PCR Template Primers Enzymes
The PCR amplification 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 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 • Anneal on opposite strands of the target sequence. • About 18 to 30 nt long and have similar G+C contents so that they anneal to their complementary sequences at similar temperatures. • Tm=2(a+t)+4(g+c): determine annealing temperature. If the primer is 18-30 nt, annealing temperature can be Tm 5oC
Tm=11x2+9x4=58oC 5’-ATTCCGATCGCTAATCGATG-3’ 3’-CACGTAAAGCGGTGATCTC-5’ Tm=9x2+10x4=58oC 5’-ATTCCGATCGCTAATCGATGGC------- TCCTGTGCA TTTCGCCACTAGAG-3’ 3’-TAAGGCTAGCGATTAGCTACCG-------AGGACACGTAAAGCGGTGATCTC-5’ 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 (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 Y= Pyrimidine N= any base R= purine
Enzymes and PCR Optimization • The most common is Taq polymerase. It has no 3’ to 5’ proofreading exonuclease activity. Accuracy is low, not good for cloning. High-accuracy DNA polymerase is available commercially. • To optimize PCR, the annealing temperature and the Mg++ concentration are varied, or the nested PCR is carried out.
Nested PCR First round primers Gene of interest Second round PCR First round PCR Second round primers
Reverse transcriptase (RT)-PCR 5‘-Cap mRNA AAA(A)n (dT)12~18 primer anneal 5‘-Cap 3‘ 5‘ AAA(A)n Reverse transcription dNTP, RT 5‘-Cap 5‘ Regular PCR AAA(A)n cDNA:mRNA hybrid
PCR mutagenesis (诱变) To introduce deletion or point mutations • Two separate PCR reactions are performed. • One PCR amplifying the 5’-portion of the insert, and the other amplifying the 3’-portion of the insert. • The point mutation/deletion mutations are located in the primers