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