molbio

• Single-Strand Conformation Polymorphism


  Widely used mutation scanning system
• Single-Strand Conformation Polymorphism


  1. Amplification

  2. Digestion

  3. Hybridization
• Electrophoresis


  • In the absence of a complementary strand, DNA/RNA forms an intrastrand duplexes forming a 3D structure/conformer (a single base pair difference in the DNA sequence can cause the conformer to fold differently)
  • Determined by the migration of the single-stranded conformers in a polyacrylamide gel
  • The speed of migration is dependent to the shape and size of the conformer
• Denaturing Gradient Gel Electrophoresis


  • Exploits differences in denaturation between a normal and mutated DNA
  • The identity of the melting domains is a function of the base sequence (mutation will likely change the melting profile)
  • Fragments are separated by using UREA and FORMAMIDE
  • As the dsDNA fragments moves through the gel, the denaturing condition increase, sequences reach their denaturing point and the complementary strands begin to denature
  • Perpendicular DGGE: the gradient increases horizontally across the gel
  • Parallel DGGE: gradient increases vertically
• Allele-Specific Oligomer Hybridization


  • Differences in TM of short sequences with one or two mismatches and those with no mismatches
  • Labeled probes (wild-type and sequence variant) matching the normal and mutated sequences are hybridized in the membrane
  • Amplification
  • Digestion
  • Electrophoresis
  • Hybridization
  • REVERSE DOT BLOT: screening individual samples for multiple allelic variants (mutant and wild-type oligonucleotides are immobilized on the solid support)
• Melt Curve Analysis


  • Sequence- and stacking-directed denaturation characteristics of DNA duplexes
  • Ethidium bromide, SYBR Green, LC green: specific for dsDNA
  • At low temperature, fluorescence increases but as the temperature rises, the duplex DNA denatures and fluorescence decreases
• Inversion Probe Assay


  • Uses a probe that hybridizes to the target sequence, the two ends flanking the potential SNP being tested
  • The probes ligates and circulates with an endonuclease and the probe is released from the target and inverts
  • The inverted probe is amplified and the amplicons are hybridized to microarray
  • Detection of SNPs in DNA
• Heteroduplex Analysis


  • Electrophoretic resolution of normal dsDNA fragments from fragments of identical length and sequence but having one base pair mismatch
  • Nonidentical dsDNA duplexes are heated to a temperature that results in complete denaturation (homoduplex wild-type, homoduplex mutant, bubble-type heteroduplex and bulge-type heteroduplex)
  • Heteroduplexes are formed when single strands are not completely complementary to hybridize each other
  • Heteroduplexes migrate differently than do homoduplexes (samples with mutations and sequence variants are characterized by the presence of extra bands with aberrant migration)
• Sequence-Specific PCR


  • AKA Amplification Refractory Mutation System
  • Commonly used to detect point mutations and other nucleotide polymorphisms
  • If there is a mismatch in the 3' end of the a primer and the DNA template, elongation by Taq polymerase will not occur (absence of amplification indicates mutation)
  • Involves the usage of primers with 3' end that falls on the nucleotide to be analyzed (specific for wild-type allele or mutant allele)
  • Detection of inherited disorders and employed in human platelet alloantigen typing
• Allelic Discrimination with Fluorogenic Probes


  • An extension of 5' nuclease PCR assay using two probes (wild type/variant sequence) with different fluors
  • Digestion occurs when a probe matches the test sequence
  • Mutant sequence labeled with VIC dye
  • Normal sequence labeled with FAM dye
• Dideoxy DNA Fingerprinting


  • Modified chain termination sequencing
  • Single ddNTP is used to generate a series of terminated fragments that are resolved in one lane of a nondenaturing polyacrylamide gel
  • A combination of Sanger sequencing, SSCP, and ddF resolves mutant and normal sequences
  • Screens only one strand of the template duplex
• Dye Terminator Incorporation


  • Utilizes fluorescently labeled ddNTPs
  • FP-TDI and SEQUENOM
  • Primers are designed to hybridize on the test sequence up to the nucleotide being tested
  • If a labeled ddNTP is incorporated in the primer, the fluorescence polarization of the dye increases
• Protein Truncation Test


  • cDNA synthesis
  • PCR
  • CHON synthesis
  • Intended for the detection of nonsense or frameshift mutations (truncating/translating terminating mutations)
  • Proteins synthesized from mutant alleles are abnormally shortened or truncated
  • Analysis of mutation in dystrophin gene and in APC gene
• RFLP-PCR


  • Mutation changes the structure of a restriction enzyme target site or changes the size of a fragment generated by restriction endonuclease
  • The region surrounding the mutation is amplified, and the mutation is detected by cutting the amplicon with restriction endonuclease
• Heteroduplex Analysis with Single-Strand Specific Nuclease


  • Cleavage of heteroduplexes at the mispaired bases with the use of S1 nuclease
  • Digested heteroduplexes yield smaller bands that can be resolved on an agarose gel
• Base Excision Scanning


  • Uracil containing amplicons yield different digestion fragments, depending on the sequence of the template
  • Utilizes dUTP and restriction endonuclease that cleave the fragment at the dU site to detect mutations affecting AT base pairs
• Nonisotopic RNAse Cleavage Assay


  The probe is hybridized to denatured target DNA. When there is a mismatch, the base or bases that have not annealed are cleaved by RNAse. The product of digestion are denatured and separated by gel electrophoresis.
• Chemical Cleavage


  • Susceptibility of specific base mismatches to modification by different chemicals
  • Uses osmium tetroxide and Hydroxylamine
• Invader Assay


  During an isothermal incubation, if the probe and the template DNA are complementary, two enzymatic cleavage reactions occur producing fluorescence.
• Friedrich Miescher


  Isolated nuclein in white blood cell nuclei
• Frederick Griffith


  Transferred killing ability between types of bacteria (S. pneumoniae: Type R to Type S)
• Bacterial transformation


  Uptake of free DNA in the environment
• Oswald Avery, Colin McLeod, Maclyn McCarty


  Discovered that DNA transmits killing ability in bacteria
• Alfred Hershey, Martha Chase


  Determined that the part of a virus that infects and replicates is its nucleic acid and not its protein
• Phoebus Levene, Erwin Chargaff, Maurice Wilkins, Rosalind Franklin


  Discovered DNA components, proportions and positions (sugar (deoxyribose and ribose), A=T; G=C, Photo 51)
• James Watson, Francis Crick


  Elucidated DNA's three-dimensional structure
• James Watson


  Coined the term molecular biology