Chapter 5

Exploring Genes and Genomes

Basic Essential Tools for Gene Exploration

1. Restriction Enzymes : Precise Sequence-Specific Cleavage of DNA

2. Blotting Techniques : Southern and Northern Blotting3. DNA Sequencing : Determination Precise Nucleotide Sequence of DNA

(human 3 billiion base-pairs)

4. Solid-Phase Synthesis of Nucleic Acids : Oligo(deoxy)nucleotide5. Polymerase Chain Reaction (PCR) : Amplification of a segment of DNA

(Nobel Prize in Chemistry 1993 Dr Kary B. Mullis, U.S.A., for his invention of the polymerase chain reaction (PCR) method)

6. Computerized Analysis : Bioinformatics

Restriction Endonuclease (Restriction Enzyme)

Rule of Naming Restriction Enzymes

• First three letters for the host organism (e.g. Hin for Haemophilus influenza)

• Next one letter for strain designation, if necessary (e.g. HinD for Haemophilus influenza D)

• Last one letter for a number, if more than one restriction enzyme have been identified from

the same species. (e.g. HinD III for the third RE found in Haemophilus influenza D)

• Found in a variety of prokaryotes.

• Foreign DNA cleavage.

• Self DNA protection by methylation

• Sequence-specific cleavage

• Hydrolysis of a phosphodiester bond

• Palindromic sequence recognition

• Staggered or even cutting

• 1978 Hamilton Smith, Daniel Nathans

PalindromicInverted repeat,

Usually4-8 base pairs

RecognitionSite for Sac I

(Streptomycesachromogenes)

Visualization of DNA Fragments by Gel Electrophoresis

Non-Denaturing Gel Electrophoresis Systems

For < 1 k bps : Polyacrylamide Gel Electrophoresis

For < 20 k bps : Agarose Gel Electrophoresis

Pulsed-Field Gel Electrophoresis (PFEG) can separate

DNA molecules in the range of million base pairs.

Denaturing Gel Electrophoresis Systems

Urea Polyacrylamide Gel can distinguish DNA fragments

differing in length just by one nucleotide out of several

hundred base pairs

EtBr (ethidium bromide) Staining of Nucleic Acids;

intercalation into base stacks; fluorescence upon UV light;

50 ng of DNA is detection limit

Ethidium bromideintercalating agent commonly used for nucleic acid staining may be a mutagen, carcinogen

Southern Blotting for DNA & Northern Blotting for RNA

DenaturationBy formaldehyde

Hybridization -rays fromradioisotope

Hybridization Probes for Southern Blots : single-stranded DNAfor Northern Blots : single-stranded mRNA

Sanger Di-Deoxy Method for DNA Sequencing

• Partial termination of DNA polymerase reaction

by including dideoxy nucleotides

• Urea Polyacrylamide Gel Electrophoresis

• Visualized by autoradiography or fluorescence

Automated Solid-Phase DNA Synthesis

Deoxyribonucleoside 3’-phosphoramidite : Incoming NucleotideDMT : Dimethoxytrityl Group ; Protect 5’ hydroxyl group of incoming nucleotideCE : -Cyanoethyl Group ; Protect 3’ phosphoryl oxygenAll bases are protected during synthesis.Phosphotriester is oxidized by iodide to make phosphodiester.Dichloroacetic acid removes DMT to generate a free 5’ hydroxyl group.Each cycle can be completed within 10 min and the efficiency is better than 98%.At the end of the synthesis, NH3 is added to remove all the protecting groups.

By HPLC, choose longest one.

Polymerase Chain Reaction (PCR) :Amplification of Specific DNA Sequences

Required Components

• Pair of primers that hybridize the target

• All four deoxyribonucleotides (dNTPs)

• A heat stable DNA polymerase

• Target sequence (template)

Cycling Reactions

• Strand Separation (Denaturation): 95

• Hybridization of Primers (Annealing): 54

• DNA Synthesis (Extension): 72

• Repeats the above three steps

Parent DNA annealing?

Theoretical PCR

Amplification Fold = 2n

(n : cycle number)

Thus,

Million Fold Amplification

after 20 Cycles, and

Billion Fold Amplification

after 30 Cycles

PCR machine PCR Application

1. Medical Diagnostics

- Detection of pathogen (bacteria

and virus)

- Detection of cancers (mutations of

ras genes)

2. Forensics

- Some genes are highly variable

within a population (human leukocyte

antigen type, HLA)

3. Molecular Evolution

- DNA is very stable and remain

intact for thousands of years or longer,

particularly when shield from air, light

and water

Vector• Autonomously replicable DNA in an

appropriate host.• Delivering vehicle for the gene of

interest into a host

Insert• DNA fragment of the gene of interest

Restriction Enzyme• To generate joinable DNA fragments

Ligase• To join the cut DNA fragments

Linker• Small DNA fragment containing

restriction enzyme sites• Can be attached to any DNA fragment

by a ligase and cut by a particular restriction enzyme to generate specifically desired cohesive ends

Plasmids

• Naturally occurring circular DNAs

acting as accessory chromosomes

(small extra chromosomes) found

in bacteria; episome, disposable

• Plasmids can be autonomously

replicated in bacteria independently

of the host chromosome.

• Plasmids utilized in molecular

cloning in the laboratory usually

contain cloning sites and selection

markers (e.g. antibiotics resistance

genes).

Useful as a cloning site

Plasmids

Evolved…

• Polylinker Region

More Cloning Sites

• Further Selection for Recombinants

X-gal Color Selection

• Stronger Replication Origin

More DNA Synthesis

Bacteriophage

arms

• Bacteriophage Inserts up to 10 kb

• Cosmid : A Circular Vector with Bacteriophage

Backbone Inserts up to 45 kb (hybrid of

plasmid/ phase)

• BAC : Bacterial Artificial Chromosome

inserts up to 300 kb

• YAC : Yeast Artificial Chromosome

inserts up to 1000 kb

Chromosome Walking

Efficient Analysis of Long Stretches of DNA

YAC

Construction of-Phage Genomic Library Cloning of a Gene

from

Genomic Library

Genomic Library Screeningwith a Radioactive Probe Containingthe Sequence of the Gene of Interest

32P-labeled probe

How to Make a Specific Probe for Library Screening ?

Make cDNA from mRNA by using Reverse Transcriptase PCR using cDNA Templates and Specific Primers for the Gene of Interest

Deduce the Possible Nucleotide Sequences from Amino Acid Sequence

Synthesize Oligonucleotides Containing Corresponding Sequences

cDNA Library Construction cDNA Library Screening

Poly(A) mRNA Isolation

using Oligo(dT) Column

Synthesis of Double-Stranded cDNA

(Total cDNA)

Linker Ligation

Cloning of Total cDNA

into Plasmid (or Bacteriophage Vectors)

Transform (or Infect) Host Bacteria

Obtain Pool of Bacteria (or Bacteriophage)

cDNA Library

• Deletions : Single Cleavage by RE

Exonuclease Digestion Ligation

• Substitutions : Oligonucleotide-Directed Mutagenesis

• Insertions : Cassette Mutagenesis

• Designer Genes : Chimeric Proteins

(cf. Immuno-Toxin Therapy for Cancer)

Mutation of DNA Generation of New Proteins

Digestion of template strand by Dpn1 enzyme

Classical Random Mutant Screening(Forward Genetics)

vs.

Generation of a Specific Mutant by Design(Reverse Genetics)

ComparativeGenomics

Evolution ?

CompleteGenome

Sequencing

Gene # ?Efficiency ?

Comprehensive Gene Expression Analysis Using Microarray

• Labeling Total cDNAs from Sample 1

with Green Fluorescence

• Labeling Total cDNAs from Sample 2

with Red Fluorescence

• Mix Two Pools of cDNAs

• Hybridize against the Gene Chip

• Variations in Gene Expression Profile

Can Be Visualized.

Breast Cancer SpecificChanges in Gene Expression

Figure out!

Animation!http://www.bio.davidson.edu/Courses/genomics/chip/chip.html

Expression of Foreign Genes in Host Cells

Expression of a Gene of Interest from Genomic DNA ???

Introns…

Splicing Apparatus Is Absent in Prokaryotes…

Introns Are Already Removed in cDNAs Made from Mature mRNAs

•+oligo T primer•High pH•Terminal transferase, add dG to 3’

+synthetic linker

Expression of Foreign Genes in Mammalian Cells

• Necessity : Post-translational Modification

Deficiency in Bacteria• Transfection

Calcium Phosphate Precipitation

Liposome-Mediated Vesicle Fusion

(cf. Stable vs. Transient Transfection)• Micro-Injection into the Nucleus• Viral Infection

DNA Virus : Adenovirus, etc. (Transient Expression)

Retro-Virus : Moloney Murine Leukemia Virus (MMLV),

Lenti-Virus, etc. (Stable Expression)

Vaccinia Virus : Viral DNA Replication in the Cytoplasm

Shut Down Host Protein Synthesis• Baculovirus : Infection into Insect Cells (Sf9),

Efficient Protein Production Factory

Transgenic Animals Can Express Exogenous Genes of Interest

Growth Hormone Deficiency DwarfismGrowth Hormone Excess Gigantism

• Metallothionein: heavy metal binding

protein with many cysteins • Growth hormone induction (500x) by

adding cadmium into drinking water• Microinjection of several hundred copies

of GH expressing plasmid into the male

pronucleus of a fertilized mouse egg

insertion into the foster female uterus• Check by southern blotting

Gene Disruption in Animal (Knock-Out Animal)

Targeted Gene Disruption

by Homologous Recombination

Myogenin(+/+) Mouse Muscle

Myogenin(-/-) Mouse Muscle

(or Markers)

Gene Knock-Out (or Knock-In) Technology Has Enabled Us to AddressIn Vivo Functions of a Gene of Interest at the Level of a Whole Organism.

Gene Expression Knockdownby RNA Interference

• Double stranded RNA cleavage by Dicer

• 21 nucleotide double stranded RNA

(siRNA : short interfering RNA)

• Recognition by RISC

(RNA Induced Silencing Complex)

• Hybridization with mRNA

• mRNA cleavage

• C.elegans, E.coli

Ti (Tumor Inducing) PlasmidsCan Deliver Foreign Genes into Plants

Crown GalPlant Tumor Caused by

Agrobacterium tumafaciens

A small synthetic plasmid carrying the gene of

interest in T-DNA fragment can be added to

Agrobacterium colonies harboring naturally

occurring Ti plasmids, then, by recombination,

recombinant Ti plasmid can be generated.

Dicot, monocot

More Methods forGene Transfer into Plants

Electroporation• Make protoplast by using cellulase• High electric pulse makes

membranes transiently permeable.• Plasmid DNA can enter the cells.• Marker selection

Gene Guns• DNA is coated onto 1-m-diameter

tungsten pellets• These microinjectiles are fired at

the target cells with a velocity

greater than 400 m s-1.GMOs in wheat, soybean, corn, and rice; most efficient way

Mutual Re-Enforcement of Protein and Nucleic Acid Chemistry