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A Week or Two of Genomics Reading: Ch9, 10, 11. At least scan it all. Focus on issues discussed in lecture. Issues covered: 1. How to sequence a genome The first time it was hard (though conceptually simple) The next millions of times its easier. 2. What we learn from a genomes sequence

3.

Human disease genes, forensics.

Technology-driven (not idea or hypothesis-driven) 1. Restriction enzymes 2. 3. 4. 5. 6. 7. Separation of DNA fragments Cloning Hybridization PCR DNA sequencing Sequence assembled

(If youve had this all, you might still learn something.)

General reasons to sequence a genome:

Identify Genes and Genomic Structure

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Hemaglobin genes and diseases (Fig 9.1, 9.20) Cells

Genome Structure

Protein

Developmental course of expression of each hemaglobin protein

More reasons to sequence a genome: Develop a new class of antibiotics DNA sequence bacterial genome Identify unique genes/proteins to bacterium

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Are those genes/proteins essential for bacterial cell? (Do genetics!) If yesmake drugs that specifically inactivate that protein.

Major Effort by Pharmaceutical Companies!

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To sequence a genome..general strategy

long DNA chromosomes..

Clone it: purify fragments & get quantity DNA sequence each fragment Assemble fragment sequences into one continuous sequence 1. 2. 3. 4. 5. 6. 7. Restriction enzymes- to make fragments Separation of DNA fragments-to identify and purify Cloning-to purify and amplify (increase quantity) Hybridization-to identify specific fragments/Southern PCR-to make 1 DNA fragment (similar to cloning) DNA sequencing- nuff said! Sequence assembled-computer nerds take over

Problem:

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Restriction enzymes: cut DNA into specific pieces

GAATTC

Cut with EcoR1

Separate/clone

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Restriction EnzymesAverage size of fragments depends on # base enzyme recognizes

Specific 10 bp - once in 106 bases Specific 16 bp -once in 4x109bases Human genome is 3 x 109 bases

16bp sequence (probably) UNIQUE!

1st step to cloning

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Need ~20kb fragments to clone (from 10000 kb chromosome)

GAATTC

EcoR1 digest

But need overlapping fragments!!! Solution: Make and clone partial digests!

GAATTC

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Sometimes you need to size separate DNA fragments

Fig 9.5

..what the gels actually look like

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Agarose gel

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Cloning DNA fragments- to purify and increase quantity for DNA sequencing

Clone- purify each from other, make 109 molecules of each!

Fig 9.7

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How to purify cloned DNA from bacterial host cell DNA..

Fig 9.9

Or, run a gel directly

Plasmid
big

small

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Libraries: Genomic vs. cDNA

In our quest to clone entire genome, make libraries from entire genomic DNA [cDNA library has sequences only from genes transcribed into mRNA (~3% of genome!)and, we cant link them up!]

Fig 9.11

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Hybridization- the key principle

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Have DNA samples (genomic DNA, for example)

Restriction enzyme digest Separate on agarose gel transfer DNA to special paper

hybridization

Total genomic DNA fragments

Wash off unbound Detect radioactive probe Gene A is on a EcoR1 fragment of this size.

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PCR- polymerase chain reaction

DNA fragment of interest primers are key

Cycles of amplification

Do this multiple times (~30)

consequences

30 cycles yields 109 molecules Enough to sequence, probe, clone, etc

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At lastDNA sequencing

Each fragment is cloned (or PCR amplified)

melt Add DNA polymerase and dATP, cGTP, dCTP, dTTP, and ddN.. & primer Radioactive label, or dye label..

The principle

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The principle

Fig 9.18

Automated with cool dyes!

What di-deoxynucleotides do

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Review of general strategy

Techniques: cloning, hybridization, PCR, DNA sequencing, assembly . Issues: Error rate.1 in 10,000 acceptable, achieved by 10 fold coverage Error due to polymorphisms~.1% sequence heterology between

homlogs, that means between any two animals.

Repeated sequences Unclonable DNA-- PCR one type of solution..

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Assembling 1000s of sequences of ~500bp each

Alignment of sequences with lots of repeats: Conceptual problem that computer guys solve (Based on strategy of sequencing ends of fragments to align) Repeat sequences*..represents the same 200bp, lets say..

**

**

GAATTC

*
Case A: Unique sequencesno problem

Case B: Repeat sequencesmake alignment problematic

Whats been sequenced

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Issues:
Error rate.1 in 10,000 acceptable, achieved by 10 fold coverage Error due to polymorphisms ~0.1% sequence heterology between homlogs, (that means between any two animals of same species). Repeated sequences Unclonable DNA-- PCR one type of solution..

Sequencing the next human genome- use primers & bulk genomic DNA!!!

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The sequences are completed (except for some gaps and unclonable DNA..)
The kinds of things weve learned.. 1. 2. ~40,000 human genes, or so Catalog of gene families & protein domains:Life is modular

3. 50% of genome repeat sequences Transposons Pseudo genes Simple repeats Segmental duplication (10KB-300kb) Other

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Classes of Repeats and DNA polymorphisms

Microsatellites

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Microsatellites: How they form, why they are so variable (high % heterozygosity) and how to detect them. Fig 11.3 Fig 11.12

One tool for forensics

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Using oligonucleotides to detect mutation/genetic makeup

Fig 11.9 and 11.10

Key: specific oligos that recognize only one allele