Download - 12 molecular techniques in radiobiology
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)