• 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