Molecular Cell Biology Fifth 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

Mullis, K.B. (1990) The unusual origin of the polymerase chain reaction. Scientific American. 262 (4) 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

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) cycles Final extension2-10mins

PCRAgarose gel electrophoresis The final productUV visualisation 3-4 hours

PCR based method

Quantitative reverse transcription polymerase chain reaction (Q-RT-PCR, or real-time -RT-PCR )

TaqMan Q-PCR

SYBR Green I Believed to be a minor groove binding dye. No probe; lower cost than the TaqMan Assay. Cannot multiplex. Detects ANY ds-DNA.

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) ≤ ≤

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 Function Sequence ESTS GENOMIC DNA Gene Disruption Homologous recombination Overexpression Anti-sense “ RNAi ” Phenotype Genetics Biochemistry Physiology

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