Genomic assays I

Cloning was the original way to amplify and store genetic seqeunces for analysis. Although largely superceded by oligonucleotide arrays and next-gen sequencing in clinical studies, it is still widely used in research settings and the Human Genome Project would not have been possible without cloning.

Cloning

PCR

The PCR is a method for exponentially amplifying DNA sequences for analysis. Reverse transcriptase PCR (RT-PCR) extneds this to amplify mRNA sequences. Finally, real-time or quantative PCR (qPCR) uses amplificatino to estimate the origianl amount of template oligonuclotide pressent and is a uniquitous assay in the laboratory. RT-PCR always refers to reverse transcriptase PCR and never to real-time PCR (qPCR).

Sanger sequencing

You should know the basic principles of Sanger sequencing, although it has been superceded by next-gen seequencing in many clinical contexts.

Oligonucleotide array

Oligonucleotide arrays are widely used for gene expression profiling (“microarrays”) as well as to identify SNPs for GWAS studies. Specificity is increased by the use of mismatch probes that indicate non-specific binding, and accuracy is increased by the use of redundancy - e.g. multiple probes for the same gene (forming a probe set), and measuring both forward and reverse strands in s SNP array.