Molecular Techniques in Radiobiology

佛教慈濟綜合醫院 放射腫瘤科 劉岱瑋

Historical Perspectives

Fred Griffith (1928) – Toxicity of Streptococcus Oswald Avery (1943) – DNA as the transforming factor Erwin Chargaff (1950) – Discover one-to-one ratio betwee

n adenine and thymine; cytosine and guanine Linus Pauling (1951) – Precise measurement of helical pol

ypeptide structure James Watson and Francis Crick (1953) – Describe the str

ucture of DNA Stanley Cohen (1972) – First recombinant DNA molecules,

pSC101

The Structure of DNA

Two antiparallel helicesDeoxyribose sugar, Phosphate group, Nucleotide

baseComplementary relationship between base pairs5’ to 3’ direction sequenceDNA self-replication during cell division

The Structure of DNA

The Structure of DNA

Hydrogen Bonds between Base Pairs

The Structures of Nucleotide in DNA and RNA

Central Dogma of Molecular Biology

Exon Exon ExonIntron IntronDNA

mRNA

Replication Transcription

Post-transcriptional modification

Protein

Translation

Post-translational modification

The Genetic Code

Restriction Endonuclease

Type II Enzyme Type III Enzyme Type I Enzyme

Protein structure Separate endonuclease and methylase

Bifunctional enzyme of 2 subunits

Bifunctional enzyme of 3 subunits

Recognition site 4-6 bp sequence,

often palindromic

5-7 bp

Asymmetric sequence

Bipartite and asymmetric

Cleavage site Same as or close to recognition site

24-26 bp downstream of recognition site

Nonspecific > 1000 bp from recognition site

Restriction & methylation

Separate reactions Simultaneous Nutually exclusive

Restriction Endonuclease

Enzyme Element of Terminology

Meaning

HindIII H Genus Haemophilus

in Species influenzae

d Strain Rd

III Third endonuclease isolated

EcoRI E Genus Escherichia

co Species coli

R Strain RY13

I First endonuclease isolated

BamHI B Genus Bacillus

am Species amylolquefaciensi

H Strain H

I First endonuclease isolated

Restriction Endonuclease

Endonuclease DNA Sequence Cleavage Products

BamH I

EcoR I

Hind III

Hae II

Pst I

Sma I

G G A T C CC C T A G G

G A A T T CC T T A A G

A A G C T TT T C G A A

G G C CC C G G

C T G C A GG A C G T C

C C C G G GG G G C C C

GC C T A G

G A T C CG

GC T T A A

A A T T CG

AT T C G A

A G C T TA

C T G C AA C G T CG

G

G GG GC CC C

C C CG G G

G G GC C C

“Sticky” ends

“Sticky” ends

“Sticky” ends

“Sticky” ends

Blunt ends

Blunt ends

VECTORS

Plasmids Bacteriophage Cosmids Yeast artificial chromosome (YAC) Viruses

Plasmids Are found in a wide variety of bacterial species Are extrachromosomal elements that behave as ac

cessary genetic units Have involved a variety of mechanisms to maintai

n a stable copy number of the plasmid in their bacterial hosts

Are dependent on the enzymes and proteins encoded by their host for their replication and transcription

Frequently contain genes coding for enzymes that are advantageous to the bacterial host

Mammalian Plasmids

Bacteriopahge was first used as a cloning vector in the early 1970s

The genome of bacteriophage is a double-stranded DNA molecules, 48502 bp in length

The DNA is carried in bacteriophage particles as a linear double-straded molecules with single-straded termini 12 nucleotides in length (cohensive termini or cos)

After entering a host bacterium, the cohensive termini associate by base pairing to form a circular molecules, then recombines into the E. coli chromosome

Bacteriophage

Mixtures of extracts prepared from bacteria infected with stains of bacteriophage carrying mutations in genes required for the assembly of bacteriophage particles – In vitro packaging (1975)

Bacteriophage can infect its host at a much higher efficiency than a plasmid

Bacteriophage can accommodates DNA inserts up to about 24,000 bp

Bacteriophage

Bacteriophage

Cosmid vectors are conventional plasmids that contain one or two copies of a small region of bacteriophage DNA – cohensive end site (cos)

The cos contains all of the cis-acting elements required for packaging of viral DNA into bacteriophage particles

Cosmids contain an antibiotic resistance gene to allow selection of infected cells

Cosmids

Cosmids

YAC are linear DNA molecules whose architecture mimics that of authentic yeast chromosome

YAC contains a centromere, telomeres and selectable markers

Most YAC libraries contain 250 kb and 400 kb of foreign DNA per clone

Yeast Artificial Chromosome (YAC)

Yeast Artificial Chromosome (YAC)

SV40 was the first viral vector for introducing foreign genes into mammalian cells

Retrovirus are ideal vectors for introducing genes into mammalian cells in a stable fasion

Adenoviral vectors Adeno-associated viral vectors Lentiviral vectors

Viral Vectors

Extraction of genomic DNA from tissue or culture cells

Partial digestion by restriction endonuclease, EcoRI Genomic DNA fragments about 40,000 bp in size a

re ligated into a cosmic vector and packaged inside becteriophage particles

The assembled bacteriophage particles are infected E. coli cells and selected by appropriate antibiotics

Genomic Library

cDNA is DNA that is complementary to the mRNA and therefore includes only the expressed genes of a particular cell

Extraction of total mRNA from tissue or culture cells

Reverse transcription to form cDNA fragments and ligated to plasmids or bacteriophage

Expression library can be screened using an antibody

cDNA Library

Host

Escherichia coli – Most widely used organism in molecular biology

Yeast – simple eukaryotes but grow as quickly and inexpensively as bacteria, yeast mutants can serve as screening method in radiobiology

Mammalian cells – - Short-term explants of cells, primary culture - Established cell line

Transformation• Alteration in cell growth pattern Increased growth to higher cell density Increase rate of growth Decrease requirement for serum growth factor Anchorage-independent growth Loss of contact inhibition -- foci• Alterations in cell surface Increase rate of transport of cell nutrients Increase secretion of protease or protease activator Increase agglutinability of glycoproteins and glycolipids Change in composition of glycoproteins, glycolipids• Alterations in intracellular components and bioc

hemical process Increase metabolic rate Increase glycosis Altered levels of cyclicnucleotides Activation or repression of certain cellular genes Change in cell cytoskeleton -- round• Tumorgenesis in nude mice

Microinjection Calcium phosphate precipitation - The most widely used method of gene transfer in vitro

- The efficiency varies markedly from one cell line to another

Liposome vector -- Complex of cationic lipid and DNA

Electroporation Gene gun Viral vectors

DNA-mediated Gene Transfer

Retrovirus Adenovirus Adeno-associated virus Herpes simplex virus Vaccinia virus Sindbis virus

Viral Vector as Gene Transfer Tools

Agarose Gel Electrophoresis

Polymerase Chain Reaction (PCR)

Enzymatic amplification of DNA fragment Forward and reward primers Heat-stable Taq DNA polymerase DNA strand denature at 94 ℃ Primers anneal to template at specific temperature DNA elongate at 72 ℃

Gene-Cloning Strategies

Choose a source of DNA - Genomic DNA or cDNA

Construction a genomic or cDNA library Screen the library to locate the gene

Functional Complementation

Hybridization

Oligonucleotide Probes

Antibody Probes

Positional Cloning

Gene AnalysesMapping

Southern Blotting

Chromosome Walking

DNA Sequence AnalysesChain-termination Method

Restriction Fragment Length Polymorphism

Restriction Fragment Length

Polymorphism (RFLP)

Single-stranded Conformation Polymorphism(SSCP)

Rely on the differences in mobility between single-stranded DNA molecules on the basis of their secondary structures in nondenaturing gels

DNA molecules of identical length but different secondary structure will migrate at different rates in nondenaturing electrophoretic gels

DNA fragments can be isolated or synthesized by performing PCR on patient DNA samples, they can then be denatured, and individual strands allowed to reanneal to themselves

SSCP can only detect about 80% of such mutations

Comparisons between

Southern, Northern and

Western Blotting

Study of Promoters:The CAT Assay

microRNA & siRNA in mammalian

MicroRNAs: small RNAs with a big role in gene regulation.He L, Hannon GJ.

microRNA: endogenous

siRNA:exogenous(cytoplasm)

(nucleus)