lecture 18 – functional genomics based on chapter 8 functional and comparative genomics copyright...

Lecture 18 –Functional GenomicsBased 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 1..84734

/organism="Homo sapiens"

/mol_type="genomic DNA"

/db_xref="taxon:9606"

/chromosome="12"

gene 1..84734

/gene="IGF1"

/note="Derived by automated computational analysis using

gene prediction method: BestRefseq."

/db_xref="GeneID:3479"

/db_xref="HGNC:5464"

/db_xref="MIM:147440"

mRNA join(1..282,4802..4958,60911..61092,78035..84734)

/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_000618.3"

/db_xref="GI:163659904"

/db_xref="GeneID:3479" /

db_xref="HGNC:5464"

/db_xref="MIM:147440"

CDS join(2100..2114,4802..4958,60911..61092,78035..78094)

/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_001104754.1"

/db_xref="GI:163659901"

/db_xref="GeneID:3479"

/db_xref="HGNC:5464"

/db_xref="MIM:147440"

Finding a Specific Clone Using a cDNA Library