dna strands can be separated under conditions which break h-bonds double-stranded molecules can be...
Post on 27-Mar-2015
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• DNA strands can be separated under conditions which break H-bonds• double-stranded molecules can be
reformed in vitro• formation of duplexes dependent on
sequence complementarity• homologous ‘probes’ are used to
detect specific nucleic acids
Nucleic Acid BlottingSouthern Blot• described by Dr. Southern (1975)• digest and electrophorese DNA• transfer to membrane• detect with probe
Northern Blot• electrophorese RNA
Dot/Slot Blot• no electrophoresis
Examples of Probes
• previously cloned genes• synthetic oligonucleotides
Northern and Southern Blots
Transfer DNA/RNA to Membrane
•Capillary Action• original method• no special apparatus• efficient, but slow
•Vacuum• special apparatus• quick and efficient
•Electrophoretic• not widely used
•Fix Membrane• ‘baking’ (heat 80o)•UV cross-link
Northern and Southern Blots
• temperature• ionic strength• chaotropic agents• probe length• probe mismatch• % GC
Factors Affecting Hybridization
Stringency and Melting TemperatureMelting Temperature (Tm)• temperature at which strands separate• can estimate Tm with formulas:
Synthetic Oligonucleotides:
Tm = 2(A + T) + 4(G + C)
Effective Tm = 81.5 + 16.6log[Na+] + 0.41(%GC) -
0.65(%formamide) – 1.4(%mismatch)*
high Tm - 15o
moderate Tm - 25o
low Tm - 35oStringency• refers to relative conditions of the hybridization
as compared to Tm
• reflects the homology between probe and target
Labeling DNA Probes• random priming• used for cloned DNA fragments
•T4 nucleotide kinase• used primarily for synthetic oligonucleotides
• (nick translation)• earlier method replaced by random priming• quality control and reproducibility problems
• (terminal transferase)• adds nucleotides to 3’ end• used primarily for generation of
homopolymer tails
Klenow = modified DNA polymerase I
Random Priming
• purify DNA fragment• kits available• use -32P-NTP or other
modified nucleotide• boil probe before
hybridization
T4 Polynucleotide Kinase
• transfers -PO4 from ATP to 5’-OH•major uses:• end-labeling (dephosphor-
ylate first if necessary)• phosphorylate synthetic
oligonucleotides•Maxim-Gilbert sequencing
Non-Radioactive DNA ProbesEnzyme-Linked Probes
• biotin/streptavidin• digoxigenin-UTP/antibody• enzyme cross-linking
RFLPRestrictionFragmentLengthPolymorphisms
• loss or gain of restriction site produces different sized DNA fragments on Southern blot• also sensitive to insertion and
deletion events • polymorphisms do not have to
be associated with the genetic locus of the probe• application depends on
enzymes and probes
Diagnosis of Genetic Diseases by RFLP
Genetic ‘Fingerprinting’• use of repetitive DNA as
probe produces complex RFLP patterns• distinguish species, strains,
individuals, etc.• can be used in diagnosis,
taxonomy, forensics, etc.
Isolates of Tuberculosis
RNA Applications• Northern blot• sizes of transcripts• gene expression information• one gene at a time
• DNA Microarrays• analyze multiple genes at one time• whole genomes
• In situ hybridization• combine with microscopy to localize
cells expressing gene
DNA Microarry (Gene Chips)• different DNA ‘probes’ are fixed onto solid
surface (glass) in array• fluorescent labeled target cDNA (mRNA)
incubated with chip• analyze 1000’s of genes
simultaneously• identification of specific
sequences (transcripts)• expression levels (mRNA
abundance)
In Situ Hybridization• incubate tissues with probe to detect cells
expressing gene of interest
• 35S-label + autoradiography• fluorescent label
= FISH
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