Review of Laboratory 3Review of Laboratory 3

• Spectrophotometric determination of DNA quantity, purity

Abs 260 nm Abs 280 nm Abs 320 nm Abs 260/Abs 280

0.05241 0.03110 0.00261 1.75

• Restriction digest, agarose gel electrophoresis

pMAB58

7577 bps

1000

2000

3000

4000

5000

6000

7000

SwaIPpuMI

AhdI

AlwNI

Asp718IKpnIApaIBsp120I

StyIBsmI

PacIBsrGI

BsaBINspV

RsrIIEco47III

BseRISexAIMluI

EcoRIXbaI

BsmIBsrGISmaI

BsaBIEcoRI

NotIMluI

BsrGIAatII++

SacII

BamHI

DraIII

Bsu36IXbaI

BspEISnaBI

ARS4/Cen6

Amp

ori

pADHNLS

ADattB1

ccdB

attB2

Ter ADH

pT7

F1

TRP1

Expected results:

Genomic DNA Uncut Plasmid DNA

Lab 3 electrophoresis resultsLab 3 electrophoresis resultsW R

• problems with ladder (λ HindIII)• incomplete digestion• what do these banding patterns represent?

Our good friend Our good friend PhaeodactylumPhaeodactylumtricornutumtricornutum

Cycle Sequencing andCycle Sequencing andBioinformatics ToolsBioinformatics Tools

Broad and Long Term ObjectiveBroad and Long Term Objective

To characterize a single clone from an To characterize a single clone from an Emiliania huxleyiEmiliania huxleyi cDNA library using cDNA library using sequence analysissequence analysis

Research PlanResearch Plan

Preparation of Competent Cells and Bacterial Transformation

Growth of Transformant and Plasmid MiniPrep

Cycle Sequencing

Sequence analysis

Today’s Laboratory Objectives Today’s Laboratory Objectives

1. To understand the theoretical basis of Sanger-type DNA sequencing

2. To learn how to analyze your E. huxleyi cDNA sequence using various web-based bioinformatics tools

What is Cycle Sequencing?What is Cycle Sequencing?

Based on the Sanger Dideoxy chain termination methodBased on the Sanger Dideoxy chain termination method

(1974) (1974)

DNA synthesis reaction whereby fluorescently labeled DNA synthesis reaction whereby fluorescently labeled dideoxynucleotides are incorportated into the newly dideoxynucleotides are incorportated into the newly replicated DNA by DNA polymerase in a primer replicated DNA by DNA polymerase in a primer extension reactionextension reaction

Separation of labeled fragments by polyacrylamide gel Separation of labeled fragments by polyacrylamide gel electrophoresiselectrophoresis

*

• Mix of dNTPs and flourescently labeled ddNTPs• ddCTP = blue, ddGTP = yellow, ddATP = green, ddTTP = red

DNA sequencing: ddNTPsDNA sequencing: ddNTPs

Incapable of phosphodiester bond formation

DNA sequencing: DNA synthesis DNA sequencing: DNA synthesis and chain terminationand chain termination

dsDNA template

single primer

chain termination byincorporation of ddNTP

Collection of labeled DNA fragments differing In length by 1 base pair

Amplitaq polymerase

96º C

50º C

60º C

Labeled DNA fragmentsLabeled DNA fragments

Reaction Products are Separated Reaction Products are Separated on a Polyacrylamide Gelon a Polyacrylamide Gel

5’-A

TG

AT

A…

……

……

……

-3’

• DNA fragments run on a 4-8% acrylamide gel allowing visualization of 1 bp size differences

• At the bottom of the gel, a stationary laser excites the passing fluorescent tags on the ddNTPs and fluorescence is measured by a detector

• software programs (e.g. phred) convert fluorescence readings to sequence

DNA sequence analysisDNA sequence analysis

ORF Finder- http://www.ncbi.nlm.nih.gov/gorf/*

BLASTN- http://www.ncbi.nlm.nih.gov/BLAST/*

BLASTX- http://www.ncbi.nlm.nih.gov/BLAST/*

Clustal W- http://www.ebi.ac.uk/clustalw/*

For more information- http://www.ncbi.nlm.nih.gov/Education/index.html

ORF finderORF finder

• ORF (Open reading frame) finder: http://www.ncbi.nlm.nih.gov/gorf/

• Why would I use it?Is there a potential protein-coding region in this sequence?Is the ORF complete (with defined start and stop codons)?Identifies ORFs in DNA sequences

• How does it work?Scans all six possible reading frames (+1, +2, +3, -1, -2, -3) for potential start codons (ATG) followed by in-frame stop codons (TAA, TGA, TAG)

Assumption: larger ORFs more likely to code for proteins

• Useful for prokaryotic genomic DNA/cDNA, eukaryotic cDNA

ORF table

Clickable

Selected ORF

BLAST selected ORF

ORF length

Translation

BLAST Database Search ToolBLAST Database Search Tool

• BLAST- Basic Local Alignment Search Tool: http://www.ncbi.nlm.nih.gov/BLAST/

• Why would I use it?Find similarity between your nucleotide/protein sequence and otherseqeuences deposited in very large (updated daily, >25 million

entry) public databases

Assumption 1: Similar DNA/protein sequences indicate descent from a common ancestral sequence (i.e. homology)

Assumption 2: Similar protein sequences indicate a similar structure and function

Program Query Sequence Database Target

BLASTN Nucleotide (both strands)

Optimized for speed not accuracy

Not good for distant homologs

Dust Filter Option (low complexity)

Nucleotide Database

BLASTX Nucleotide translated 6 frames

Less sensitive to sequence errors and mismatches

Useful for preliminary data/EST

Dust Filter Option

Protein Database

BLASTP Protein Protein Database

The BLAST FamilyThe BLAST Family

How does it work? How does it work? The BLAST AlgorithmThe BLAST Algorithm

Performs similarity search between your query sequence and aPerforms similarity search between your query sequence and anucleotide/protein database.nucleotide/protein database.

Pairwise alignment (two sequences compared)Pairwise alignment (two sequences compared)

Query sequence is split into small fragments (words) of defaultQuery sequence is split into small fragments (words) of defaultsizes of 11 bp or 3 amino acidssizes of 11 bp or 3 amino acids

Words are compared to every sequence in the target databaseWords are compared to every sequence in the target database(sliding window)(sliding window)

ATTGTACCGTA…GCGAT-ATACAGTTTTA…

How does it work?How does it work?The BLAST AlgorithmThe BLAST Algorithm

• Every window is scored using a scoring matrix, e.g. Every window is scored using a scoring matrix, e.g. BLOSUM62 or PAMBLOSUM62 or PAM

Nucleotide matrix- Match = +3, Substitution = -1, Gap = -5Nucleotide matrix- Match = +3, Substitution = -1, Gap = -5 Amino acid matrix- more complex relationships betweenAmino acid matrix- more complex relationships between amino acid substitutionsamino acid substitutions

• Goal to identify HSP’s (High Scoring Segment Pairs), Goal to identify HSP’s (High Scoring Segment Pairs), examine adjacent regions of sequence (Local alignment)examine adjacent regions of sequence (Local alignment)

• total alignment score is subjected to statistical analysis to total alignment score is subjected to statistical analysis to calculate the significance vs. random chance of the scorecalculate the significance vs. random chance of the score

ATTGTACCGTA…GCGAT-ATACAGTTTTA…

BLASTX ResultsBLASTX Results

Interpreting BLAST ResultsInterpreting BLAST Results

•Length

•E-Value (<1 x 10-3)

•Bit Score (>30)

•Identity (>25% amino acid i.d.)

•Positives

Clustal W: multiple sequence Clustal W: multiple sequence alignmentsalignments

ClustalW: http://www.ebi.ac.uk/clustalw/

Allows comparison of more Allows comparison of more than two sequences, with than two sequences, with identical and similar residues identical and similar residues aligned in columnsaligned in columns

Assumption 1: similar Assumption 1: similar sequences are structurally, sequences are structurally, functionally, and evolutionarily functionally, and evolutionarily relatedrelated

Assumption 2: specific Assumption 2: specific conserved residues (a.a. or conserved residues (a.a. or nt) are functionally importantnt) are functionally important

Why would I do a multiple Why would I do a multiple sequence alignment?sequence alignment?

Phylogenetic comparisons (more similar Phylogenetic comparisons (more similar sequences are more closely related)sequences are more closely related)

Identify highly conserved amino Identify highly conserved amino acid/nucleotide sequences (may be critical acid/nucleotide sequences (may be critical for function)for function)

Characterize gene familyCharacterize gene family Identify conserved regions for the design Identify conserved regions for the design

of PCR primersof PCR primers

How does Clustal W work?How does Clustal W work? True multiple sequence alignment is extremely True multiple sequence alignment is extremely

computationally taxingcomputationally taxing Clustal W strategy: progressive pairwise Clustal W strategy: progressive pairwise

alignmentsalignments

A+B C+D E+F

Consensus1

Consensus2

Consensus3

+

Consensus4 + Final alignment

Pairwise scores

Alignment editor

Colored alignment

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