Lecture 18 – Functional Genomics Based on chapter 8 Functional and Comparative Genomics Copyright © 2010 Pearson Education Inc.

1. Questions you should be able to answer from today’s lecture. 1.How can we compare genomic DNA sequences? 2.How can we determine whether a gene is, or is not, transcribed in a particular sample? 3.How can we determine the abundance of a particular RNA in a sample? 4.How can we identify proteins that interact? 2. Molecular Analysis of Cloned DNA Cloned DNA is used in many types of experiments. Two examples are: –Southern blotting. –Northern blotting.

1 - RNA Expression Analysis – Determining Genomewide RNA Expression Levels Northern blotting – Review RT-PCR – Review Real-time PCR – Review Genomewide RNA expression analysis Types of microarrays Making microarrays Hybridization to microarrays

Slide 3 - Southern Blot Analysis of Sequences in the Genome

2 - Northern Blot Analysis

Slide 5 - The Wide Range of Uses of the Polymerase Chain Reaction (PCR) Advantages and Limitations of PCR  PCR is more sensitive faster than cloning, but there are limitations:  Specific primers require that sequence information be known.  No more than about 40 kb can be amplified.  Taq polymerase does not proofread, meaning that mismatches go uncorrected. Alternative polymerases such as Vent polymerase do proofread, decreasing errors.  The sensitivity can result in amplification of contaminating sequences, a special hazard in forensic applications.

6. Applications of PCR Applications of PCR include:  Amplifying DNA for cloning.  Amplifying DNA from genomic DNA for sequencing without cloning.  Mapping DNA segments.  Disease diagnosis.  Subcloning segments of cloned DNA.  Individual genes may be amplified from a cloned multigene DNA fragment.  Complementation is used to determine functions of each gene.  Forensics (the analysis of legal evidence) in samples including hair, blood, or semen.

4 - RT-PCR and mRNA Quantification 1. Isolate mRNA 2. Reverse transcribe mRNA (make a DNA copy of each mRNA) 3. PCR amplify the first strand reverse transcribed mRNAs 4. Agarose Electrophoresis of samples

5 - Real-time PCR - Review  Real-time PCR is a form of reverse transcription RCR where the method of analysis involves continuous monitoring of the PCR product formed.

11. Alternative Pre-mRNA Splicing: P Element Transposition in Drosophila

Synthesis of cDNAs

Building cDNA Libraries

Using a cDNA Library to Annotate Genes FEATURES Location/Qualifiers source /organism="Homo sapiens" /mol_type="genomic DNA" /db_xref="taxon:9606"9606 /chromosome="12" gene /gene="IGF1" /note="Derived by automated computational analysis using gene prediction method: BestRefseq." /db_xref="GeneID:3479"3479 /db_xref="HGNC:5464"5464 /db_xref="MIM:147440" mRNA join(1..282, , , ) /gene="IGF1" /product="insulin-like growth factor 1 (somatomedin C), transcript variant 4" /note="Derived by automated computational analysis using gene prediction method: BestRefseq.“ /transcript_id="NM_ "NM_ /db_xref="GI: " /db_xref="GeneID:3479" /3479 db_xref="HGNC:5464"5464 /db_xref="MIM:147440" CDS join( , , , ) /gene="IGF1" /note="isoform 2 preproprotein is encoded by transcript variant 2; Derived by automated computational analysis using gene prediction method: BestRefseq." /codon_start=1 /product="insulin-like growth factor 1 isoform 2 preproprotein" /protein_id="NP_ "NP_ /db_xref="GI: " /db_xref="GeneID:3479"3479 /db_xref="HGNC:5464"5464 /db_xref="MIM:147440"147440

Finding a Specific Clone Using a cDNA Library