Pillars - Genotyping

PCR

• Polymerase Chain Reaction

• 1. Denaturation: DNA strands heated, separate

• 2. Annealing: DNA cooled, DNA primers attach to DNA template

• 3. Extension: Temperature raised, Taq polymerase makes new DNA strand based on template

• requires some knowledge of sequence to make forward primer and backward primer

  • more G-C pairs in primer increases strength of binding, can bind at higher annealing temps to reduce non-specific binding

  • need both forward and reverse to amplify bc otherwise template won’t have anything to bind to

Nucleic Acid Techniques

• PCR

• RT-PCR

• Genetic Mapping by DNA markers

• Rapid Amplification of cDNA ends (RACE)

• Southern, northern, in situ hydridizations

• Genetic Engineering and Gene Cloning

Dominance (molecular markers)

• molecular marker reports diversity (mutation) at only one allele for any particular gene or locus

• primer made to bind to particular mutation: if no mutation, primer won’t bind and there will be no PCR product

Co-dominance (molecular markers)

molecular marker reports diversity (mutation) at both alleles for any particular gene or locus

can identify hetero vs homozygous

RFLP

• Restriction Fragment Length Polymorphism

• Co-dominant marker

• first DNA profiling tech used for wide application

• digest genomic DNA with restriction enzymes and hybridize with radioactive nucleotide labeled primer

  • requires sequence info

• will cut however many times sequence is found

• cheaper, time consuming

• high reproducibility

RAPD

• Random Amplification of Polymorphic DNA

• PCR of genomic DNA with single RAPD primer

  • decamer (10 nucleotide) of arbitrary sequence as primer

  • do not need sequence info

• easy, low reproducibility

  • not very accurate

• Dominant marker: heterozygous will show similar banding pattern with wild type

AFLP

• amplified fragment length polymorphisms

• combination of RFLP and RAPD

• dominant marker

• add restriction enzyme, cut genome, then do PCR

• easier than RFLP, highly reproducible

• use cDNA-AFLP to identify genes involved in disease

• clone novel genes by PAGE elution with specific primers

• does not need sequence info

Microsatellites

• SSR: Simple Sequence Repeat

• ISSR: Inter-Simple Sequence Repeat

• Codominant

• These are short, repeating sequences of DNA found throughout the genome, and variations in the number of repeats at specific locations (loci) can be used to differentiate individuals or populations

SSR DNA Length Polymorphisms

• design primer on either side of simple sequence repeat and amplify the SSR

• may be anywhere in genome, many in exons

• highly reproducible, good tech for gene mapping

ISSR

• genome region between microsatellite loci

• complementary sequences to two neighboring microsatellites are used as PCR primers

• variable region between them gets amplified

• easier than SSR

  • don’t need to know exact sequence

• highly reproducible

Molecular Beacons

• molecular beacon hs quencher and fluorophore together

• quencher makes fluorophore not glow

• when primer with molecular beacon binds to template, fluorophore separates from quencher

• intensity of light shows how many templates bind to template

Real Time PCR

• the formation of amplification product is detected at each cycle

• allow you to see how many mutation is in sample