Avidin a chicken egg white protein or streptoavidin a

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avidin (a chicken egg white protein) or streptoavidin (a bacterial  analog of avidin) added biotin-labeled enzyme (e.g., alkaline phosphatase or peroxidase);  biotin is vitamin B 7 – binds very tightly,  K d   = 10 -15  M Chromogenic or chemiluminescent substrate added
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Chemiluminescent detection of target DNA 4 sites/molecule
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Other Advantages of Non-radioactive Detection  Systems biotin-labeled DNA stable >1 y, RT device: high sensitivity detection options  X-ray film luminometer  color change, < a few hr  chemiluminescence (emits light) more sensitive than  chromogenic  dyes detection system of choice  
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Use of fluorescent dyes that are attached to primers for  detecting amplified PCR products
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Molecular Beacons 25-nt long middle 15 nt complementary to the target DNA 5 nt at each end complementary to each other 5’ end, a fluorescent chromophore ( fluorophore ) 3’ end, a nonfluorescent chromophore ( quencher ) quencher absorbs energy emitted by the fluorophore before it  fluoresces
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Hybridization of molecular beacon probe to target DNA
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Fig. 9.10 Molecular beacon probes with different fluorophores
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Specificity perfect match in all 15 nt hybridizes  1 mismatch does not  hybridize
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Use of two different molecular beacon probes to distinguish  between different genotypes H
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DNA Fingerprinting Distinguishes individuals by DNA analysis Uses of DNA typing links suspects to the crime scene wrongly convicted? paternity victims
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Two  Approaches A set of probes to  hybridize  undegraded  minisatellite DNA restriction digest  agarose gel electrophoresis,  transblot onto nylon membrane, hybridize, strip, hybridize A set of primers to  amplify  minute samples or  degraded DNA
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Southern blot  of a forensic DNA sample
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Human Mini-satellite DNAs throughout the human genome tandemly repeated sequences 9 to 40 bp each repeat, 10-30 repeats children inherit one set of minisatellite DNA  sequences from each parent exhibit extensive variability One in 10 5  to 10 8   having the same fingerprint
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Human mini-satellite DNA 9 nt repeat 5 6 7
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Random  Amplified  Polymeric DNA ( RAPD ) 9 to 10 bp primers No palindromic sequences G+C content 50 to 80% Anneals to many sites if a primer anneals to opposite strands 100-3,000 bp apart a fragment will be amplified Change of one bp results in change of amplification  pattern
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Binding of a single random oligonucleotide (arrow) to the  chromosomal DNA
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PAGE of  PCR -amplified plant DNA with Ethidium Bromide staining of bands
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Advantages of  RAPD universal set of primers
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  • Fall '08
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  • DNA, Trigraph, multiple myeloma, ro te, rid iza tio

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Christopher Reinemann
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