Methods in Cell Biology Cont. Sept. 24,

Science Bomb 2 Unc-22: encodes a myofilament in C. elegans

Genomic DNA Sequencing mRNA Total RNA miRNA Genomic DNA Captured/PCR/IP DNA RNA Whole Genome Assembly Copy Number Variation Genome rearrangements Chromatin structure Regulation of Gene Expression Splice Site Monitoring/Discovery SNV/Indel Identification Gene Discovery Any Species Bisulfite treated Expression Profiling 3

Targeted Sequencing: PCR mRNA Total RNA miRNA Genomic DNA Captured/PCR/IP DNA RNA Whole Genome Assembly Copy Number Variation Genome rearrangements Chromatin structure Regulation of Gene Expression Splice Site Monitoring/Discovery SNV/Indel Identification Gene Discovery Any Species Bisulfite treated Expression Profiling 4

5 PCR

6

Targeted Sequencing: PCR Millions of reads SNV/Indel Discovery 7

Genomic DNA Sequencing mRNA Total RNA miRNA Genomic DNA Captured/PCR/IP DNA RNA Whole Genome Assembly Copy Number Variation Genome rearrangements Chromatin structure Regulation of Gene Expression Splice Site Monitoring/Discovery SNV/Indel Identification Gene Discovery Any Species Bisulfite treated Expression Profiling 8

Applications of PCR 1) Cloning a gene encoding a known protein Primers can be designed from sequence of amino acids or gene sequence. 2) Amplifying 'old DNA' Amplifying DNA sequences from museum material or fossils - look at evolution of gene sequences (Molecular evolution studies) 3) Amplifying cloned DNA from vectors Convenient way of checking the inserts - amplified DNA can be analysed by electrophoresis, Southern blottingSouthern blotting 4) Creating mutations in cloned genes 5) Detecting bacterial or viral infection More sensitive than conventional diagnostic techniques (culturing samples from patients or using antibodies to detect the presence of microorganisms and viruses) 6) Cancer Detecting mutations that occur in cancer and monitoring cancer therapy. Determining if a patient is free of malignant cells PCR is one of the most versatile techniques invented, and has so many applications that this list could go on for quite some time. 9

Extensions of PCR 1) RT-PCR (reverse transcription PCR) – Uses reverse transcriptase 2) qPCR (quantitative PCR) 3) qRT-PCR (quantitative RT-PCR) – Combine methods above – Outstanding ability to quantify levels of mRNA RNA template5’3’ 10

Targeted Sequencing: Captured mRNA Total RNA miRNA Genomic DNA Captured/PCR/IP DNA RNA Whole Genome Assembly Copy Number Variation Genome rearrangements Chromatin structure Regulation of Gene Expression Splice Site Monitoring/Discovery SNV/Indel Identification Gene Discovery Any Species Bisulfite treated Expression Profiling 11

12 Targeted Sequencing: Captured

Genomic DNA Sequencing mRNA Total RNA miRNA Genomic DNA Captured/PCR/IP DNA RNA Whole Genome Assembly Copy Number Variation Genome rearrangements Chromatin structure Regulation of Gene Expression Splice Site Monitoring/Discovery SNV/Indel Identification Gene Discovery Any Species Bisulfite treated Expression Profiling 13

Genomic DNA Sequencing mRNA Total RNA miRNA Genomic DNA Captured/PCR/IP DNA RNA Whole Genome Assembly Copy Number Variation Genome rearrangements Chromatin structure Regulation of Gene Expression Splice Site Monitoring/Discovery SNV/Indel Identification Gene Discovery Any Species Bisulfite treated Expression Profiling 14

15 Bisulfite Sequencing ACGTGACGT ATGTGACGT me AUGTGACGT me bisulfite PCR

Genomic DNA Sequencing mRNA Total RNA miRNA Genomic DNA Captured/PCR/IP DNA RNA Whole Genome Assembly Copy Number Variation Genome rearrangements Chromatin structure Regulation of Gene Expression Splice Site Monitoring/Discovery SNV/Indel Identification Gene Discovery Any Species Bisulfite treated Expression Profiling 16

RNA-Seq mRNA Total RNA miRNA Genomic DNA Captured/PCR/IP DNA RNA Whole Genome Assembly Copy Number Variation Genome rearrangements Chromatin structure Regulation of Gene Expression Splice Site Monitoring/Discovery SNV/Indel Identification Gene Discovery Any Species Bisulfite treated Expression Profiling 17

RNA-Seq total RNA double stranded DNA polyA+ RNA column purify PCR Blunt-end ligate adaptors Fragment RNA Random DNA primers (NNNNNN) 18

RNA-Seq mRNA Total RNA miRNA Genomic DNA Captured/PCR/IP DNA RNA Whole Genome Assembly Copy Number Variation Genome rearrangements Chromatin structure Regulation of Gene Expression Splice Site Monitoring/Discovery SNV/Indel Identification Gene Discovery Any Species Bisulfite treated Expression Profiling 19

Gel Electrophoresis 20 purify small RNAs

Northerns/Southerns 21 Westerns: electrophorese proteins, use antibody for detection

In situ hybridization: measuring gene expression

Sanger Sequencing 23

Sanger Sequencing 24

Sanger Sequencing 25

Methods to perturb the cell All methods covered up to this points are designed to quantify Methods for perturbing cell activity needed to test for causality (e.g. defective gene X causes higher expression of gene Y, need to mutate gene X, then measure gene Y) – RNAi – Reporter genes – Genetic engineering Cloning Transient plasmid expression Genomic integration Knockout genes – Mutational screening 26

RNA interference (RNAi) 27 Get dsRNA into cells by injection, on a plasmid, or a custom virus

Reporter Genes: green fluorescent protein (GFP)

GFP mice

GFP actin filaments