molecular cell biology fifth edition chapter 9: molecular genetic techniques and genomics copyright...
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Molecular Cell BiologyFifth Edition
Chapter 9:Molecular Genetic Techniques
and Genomics
Copyright © 2004 by W. H. Freeman & Company
Harvey Lodish • Arnold Berk • Paul Matsudaira • Chris A. Kaiser • Monty Krieger • Matthew P. Scott • Lawrence Zipursky •
James Darnell
You are going to study the effect of a factor or drug to a human cell line and identify the up-regulation or down-regulation genes.
When your advisor assigns this project to you, how do you approach this problem?
Problems
1. What kind of strategy (methods) to approach?
2. What kind of techniques, equipments are you going to use?
3. How can you confirm your gene up- or down-expressed in the cell line?
4. How can you clone these genes?
Issues to be focus (I)
5. What kinds of vector you can use?
6. What kinds of library are you going to use, if any. If not, please give your reason?
7. How to study the function of the cloned gene?
8. What methods can you carry out to determine the cloned gene has mutation(s)?
Issues to be focus (II)
1 What kind of strategy (methods) to approach?
2. What kind of techniques, equipments are you going to use?
3. How can you confirm your gene aberrantly or normally expressed in the cell line?
4.How can you clone this gene?
Issues to be focus (I)
1 What kind of strategy (methods) to approach?
2. What kind of techniques, equipments are you going to use?
3. How can you confirm your gene aberrantly or normally expressed in the cell line?
4.How can you clone this gene?
Issues to be focus (I)
Blotting Techniques Northern Blotting
RNA run on a gel, probed with complementary DNA or RNA probe
for studying the (mRNA) expression of genes can determine size of mRNA
Western Blotting Protein run on a gel, probed with specific
antibody for studying expression, size of proteins
Southern, Northern, Western Blotting Compared
http://www.nobel.se/chemistry/laureates/1993/mullis-autobio.html
Mullis, K.B. (1990) The unusual origin of the polymerase chain reaction. Scientific American. 262 (4) 56-65.
devised by Kary Mullis c1983
POLYMERASE CHAIN REACTION - PCR
A 'licence' to do molecular biology
A key central technique that has revolutionised molecular and consequently cell biology
Polymerase Chain Reaction (PCR)
•PCR is an in vitro technique for the amplification of a region of DNA which lies between two regions of known sequence.
•PCR amplification is achieved by using oligonucleotide primers.
•These are typically short, single stranded oligonucleotides which are complementary to the outer regions of known sequence.
The steps of:
1. Template denaturation
2. Primer annealing
3. Primer extension
http://wine1.sb.fsu.edu/bch5425/lect22/lect22.htm
CYCLING PARAMETERS
Denaturation; 93°C - 95°C
30 secs – 1min
Annealing; 37°C - 65°C
30 secs – 1min depends on the duplex
Extension; 72°C
1min (+ 30secs per 500bp DNA)
25-35 cycles
Final extension 2-10mins
PCR Agarose gel electrophoresis
The final product UV visualisation
3-4 hours
PCR based method
Quantitative reverse transcription polymerase chain reaction (Q-RT-PCR, or real-time -RT-PCR )
TaqMan Q-PCR
Real-time Quantitative PCR (Applied Biosystems: 7900HT)
1 What kind of strategy (methods) to approach?
2. What kind of core facility (or techniques, equipments) are you going to use?
3. How can you confirm your gene aberrantly or normally expressed in the cell line?
4.How can you clone this gene?
Issues to be focus (I)
5. What kinds of vector you can use?
6. What kinds of library are you going to use, if any. If not, please give your reason?
7. How to study the function of the cloned gene?
8. What methods can you carry out to determine the cloned gene has mutation?
Issues to be focus (II)
Clone by RT-PCR
Different Cloning Vectors for Different Applications
Sizes of inserted DNA commonly obtained with different cloning vectors
Cloning Vector
Standard high copy number plasmid
Bacteriophage
Cosmid
Bacteriophage P1
PAC (P1 Artificial Chromosome)
BAC (Bacterial Artificial Chromosome)
YAC (Yeast Artificial Chromosome)
Size of insert (kb)
≤ 10
9-23
30-44
70-100
130-150
≤ 300
0.2-2000
Plasmid-Based Cloning Vectors Naturally occurring plasmids must be modified in
order to be used as cloning vectors
Insertion of a multiple cloning site (MCS) polylinker ≈ 30bp synthetic sequence with recognition sites
for several common restriction enzymes .
Insertion of an antibiotics resistance gene for selection in transformed bacterial cells growing in selective medium
Plasmid vectors Plasmid vector is: a small, self-reproducing piece of circular
DNA found outside of the chromosome. the simplest bacterial vector that is used as a
vehicle to carry foreign DNA sequences into E. coli or another host cell.
PLASMIDS• CIRCULAR DNA MOLECULES
• AUTONOMOUSLY REPLICATING
• SELECTABLE MARKER eg AMP RES
• POLYLINKER OR MCS
= MULTIPLE CLONING SITE
eg pBR322, pUC18
Properties of plasmid vectors
Smaller plasmid vectors are preferred for many reasons:
1)the efficiency of transformation is inversely related to the size of the plasmid.
The size becomes a limiting factor when the plasmid exceeds 15 kb.
Properties of plasmid vectors
Smaller plasmids can accommodate larger segments of foreign DNA before the efficiency begins to deteriorate.
2) Larger plasmids are more difficult to characterize by restriction mapping.
Properties of plasmid vectors
3) The yield of foreign DNA is reduced with larger plasmids because these plasmids replicate to lower copy numbers.
Selection for Recombinant Bacterial Clones
The vector used must harbour a suitable marker gene whose activity in the target cell can facilitate the identification of cells carrying it 1. Antibiotic resistance-conferring
markers Unmodified (wild-type) host must
be sensitive the the chosen antibiotic
Ampicillin Tetracycline Chloramphenicol
PHAGE BACTERIAL VIRUS MAINLY Larger capacity of insert than
PLASMIDS
5. What kinds of vector you can use?
6. What kinds of library are you going to use, if any. If not, please give your reason?
7. How to study the function of the cloned gene?
8. What methods can you carry out to determine the cloned gene has mutation?
Issues to be focus (II)
TO CLONE A GENE from library
MAKE A LIBRARY IN A VECTOR LIBRARY = REPRESENTATIVE
COLLECTION OF DNA FRAGMENTS
SCREENING
1) COMPLEMENTATION/DNA HYBRIDISATION
2) ANTIBODY/EXPRESSION LIBRARY
5. What kinds of vector you can use?
6. What kinds of library are you going to use, if any. If not, please give your reason?
7. How to study the function of the cloned gene?
8. What methods can you carry out to determine the cloned gene has mutation?
Issues to be focus (II)
Reverse Genetics
FunctionSequence
ESTSGENOMIC DNA
Gene Disruption
Homologous recombinationOverexpression
Anti-sense“RNAi”
Phenotype
GeneticsBiochemistryPhysiology
How to study gene function?
Genetic
Gene knock-out
How to study gene function?
Epigenetic
antisense oligonucleotide RNAi
Formation of antisense RNA blocks translation
RNA interference (RNAi)
• A phenomenon in which small interfering RNA (siRNA) specifically suppresses the expression of target protein by degrading the target mRNA.
Transgenic mice
Gene overexpression
Knockout
Conditional Knockout
5. What kinds of vector you can use?
6. What kinds of library are you going to use, if any. If not, please give your reason?
7. How to study the function of the cloned gene?
8. What methods can you carry out to determine the cloned gene has mutation?
Issues to be focus (II)
DNA SEQUENCING TWO METHODS 1The chemical degradation method
Maxam & Gilbert
2 The dideoxy method – also called the chain termination or Sanger method invented by Sanger, Smith & Coulson in
early 70s
To Perform Sequencing PRIMER/TEMPLATE MIX DISTRIBUTED TO
FOUR TUBES 4 dNTPs PLUS DNA POLYMERASE A RADIOACTIVE dNTP IN ONE OF FOUR TUBES ADD ddA IN A SECOND ADD ddC THIRD ddG FOURTH ddT
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