Dot plot - PowerPoint Presentation

Slide1

Dot plot

Daniel SvozilSlide2

Software choice

source: Bioinformatics for DummiesSlide3

Dotlet

Learn by example – use the sequence from the Repeated domainsSlide4

In

this case, the darker the pixel, the lower the

score.

There will be a large number of pixels with low scores and only a few ones with high scores.Tune the grayscale in order to make the background noise (low scores) disappear and the similar regions stand out more clearly.To do this, use the histogram window.This represents the frequency of each score, over all the pixels, on linear (blue)

and

logarithmic (purple)

scales.

lowest

possible score on the left and the highest

on the rightIf the sequence has some similarity, there will

be a smaller peak of higher scores.

Semi-logarithmic plot makes it even more visible.Slide5

With the scrollbars below and above the histogram, respectively, bring the lower threshold just past the first peak, and the higher threshold just past the second

peak.

Now

, the background noise has disappeared from the dots window, and the similar regions stand out more clearly. Slide6

Well matching residues – blue.

The cursor can also be moved with the keyboard with the arrow keys, and with '<' (up left), '>' (down right), '[' (up right), and ']' (down left).

Now play with all sequences in Dotlet exampes section, read the comment and try to understand:

http://myhits.isb-sib.ch/util/dotlet/doc/dotlet_examples.htmlSlide7

Getting the right window size

Long windows = clean plots.

The size of a window should be within

the same range as the size of the elements you’re looking for. For instance, if you’re looking for conserved domains in proteins, a size of 50 amino acids or higher is appropriate.Shorter windows are more sensitive but bring some noise with them.Start

with a large

window and

narrow it a little until the signal

you’re looking

for appears.Slide8

More of Dotlet

What is the UniProtKB database?

What

are the UniProtKB/Swiss-Prot and UniProtKB/TrEMBL? What is the difference between them?Using Dotlet, compare following two Uniprot sequences: P05049 (1st sequence) and P08246 (2nd sequence).Are these sequences homologous?What is the function of P05049?P05049 is a serine protease. Would you run a wet lab experiment to check the protease activity of P08246?

You should check if these two sequences are homologous in the serine protease region. Do you see some homologous regions on the dotplot?Slide9

Working with a single DNA sequenceSlide10

Removing vector sequences

Contamination from your own vector sequence (as a responsible scientist, you’re expected to have this information) – you may search for the vector sequence you expect

Cross-contamination by somebody else’s vector – search not only for the sequence you expect, but also for other possible vector sequences.

Before working with your DNA sequence, you should always clean it with e.g. NCBI VecScreen http://www.ncbi.nlm.nih.gov/VecScreen/VecScreen.html

Basically, it performs a blastn search against UniVec – database of vector sequencesSlide11

VecScreen

Sources of contamination -

http://

www.ncbi.nlm.nih.gov/VecScreen/contam.html Trysequence1.txt"No significant similarity found“ - a good news, indicates that the sequence does not contain any vector contamination sequences.sequence2.txt Slide12

the query sequence matches three vector sequences

Let’s say we know

the vector used

for

cloning: pCR2.1-TOPO.

Which sequence would you remove?

Remove this sequence and check the results on the cleaned sequence.Slide13

Clean sequence3.txt. What is the result?

Such a sequence is generally considered as the esult of a chimeric clone – i.e. clone consisting two sequences.

In this case, throwing it away is the safest thing to do!

In sequence4.txt is a sequence you cloned in the vector pUC19. Is it contaminated? How would you clean it?VecScreen reports a strong match with the lactose operon genes from E. coli. Not from pUC19!However, this is ok as most commercial vectors are derived from the same initial natural plasmid and E. coli constructs. Their sequences are identical, and UniVec matches are reported in the ordedr they appear in the database.

http://

www.ncbi.nlm.nih.gov/VecScreen/Interpretation.html

Slide14

Restriction map

It is possible to cut DNA sequences using restriction

enzymes.

Each type of restriction enzyme recognizes and cuts a different sequence:EcoR1: GAATTCBamH1: GGATCCThere are more than 900 different restriction enzymes, each with a different specificityThe restriction map is the list of all potential cleavage sites in a DNA moleculeSlide15

Restriction map

To compute a restriction map is not that difficult. All you need to do is to look for exact matches of a given restriction-enzyme site within your sequence.

Enzymes and sites are in the REBASE database -

http://rebase.neb.com Nebcutter - http://tools.neb.com/NEBcutter2/Webcutter - http://rna.lundberg.gu.se/cutter2

/

VIRS -

http://

bis.zju.edu.cn/virs/index.html

Try to construct a restriction map of the sequence5.fasta.Slide16

PCR primer design

DNA polymerase

needs a template

can only extend an existing piece of DNA (primer)always moves in the 5’ → 3’ directionSteps of PCRdenaturation – 94°Cannealing – 60°Cextension – 72°C

Heat

CoolSlide17

PCR primer design

DNA polymerase

needs a template

can only extend an existing piece of DNA (primer)always moves in the 5’ → 3’ directionSteps of PCRdenaturation – 94°Cannealing – 60°Cextension – 72°CSlide18

PCR primer design

DNA polymerase

needs a template

can only extend an existing piece of DNA (primer)

always moves in the 5’ → 3’ direction

Steps of PCR

denaturation – 94°C

annealing – 60°C

extension

– 72°CSlide19

Primers

primer sequence

Need

to be chosen to uniquely select for a region of DNA, avoiding the possibility of mishybridization to a similar sequence nearby.primer length18-30 bp (18-22 optimum)primers longer tha 30 bps are not specific enoughprimer melting temperature Tm

Temperature at which DNA duplex dissociates to become single stranded

Pairs of primers should have similar melting temperatures since annealing in a PCR occurs for

both

simultaneously.

Itakura’s empirical rule (quick and dirty, works “well” for temperatures 45°C-70°C, Wallace temperature)

 Slide20

Primers

primer melting temperature T

m

more accurate estimations – nearest neighbor modelOligoCalc - http://www.basic.northwestern.edu/biotools/oligocalc.html primer annealing temperature T

a

depends on the length and composition of primers

Rychlik formula

Too

low

T

a

– one or both primers will anneal to sequences other than true target, as internal single-base mismatches or partial annealing may

be

tolerated. This can lead to nonspecific amplification and will consequently reduce the yield of the

desired

product.

Too high

T

a

–

may

yield little product, as the likelihood of primer annealing is reduced.

 Slide21

Primers

GC content

Primers with a 40-60% GC content ensure stable binding of

primer/template.The presence of G or C bases at the 3′ end of primers (GC clamp) helps to promote correct binding at the 3′ end due to the stronger hydrogen bonding of G and C bases.However, strings of G and of C can form internal, non-Watson-Crick base pairs that disrupt stable primer binding. Generally, sequences containing more than three repeats of G or of C in sequence should be avoided in the first five bases from the 3′ end of the primer.

A short run of G’s at or near the 5′ end of a primer will not disrupt stable binding because the 5′ positioning does not lead to involvement in disruptive secondary structures

.

It

is best to select primers with a random base distribution.Slide22

Primers

no secondary structures

Presence of the primer secondary structures produced by intermolecular or intramolecular interactions can lead to poor or no yield of the product

. e,g, hairpins, self dimers, cross dimersIt is desirable to design specific primer pairs which do not assume secondary structures during the

reaction.

AutoDimer

-

screens

primers for primer-dimer and hairpins http://

www.cstl.nist.gov/div831/strbase/AutoDimerHomepage/AutoDimerProgramHomepage.htm

source: http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.htmlSlide23

PCR Primer Design

Pick some sequence from NCBI nucleotide (<1000 bp) and play with the primer design tool Primer3

– from

http://biotools.umassmed.eduAfter you’ve got your primers, you must verify they will not hybridize anywhere except you intend them to hybridize.e.g. primer sequences are not outside the gene you’re interested inor primers do not resemble a frequent repeats in DNATechnique for avoiding this problem: BLAST searches against the vector sequences, the genome sequences, their most common repeats.Slide24

PCR Primer Design

PrimerBLAST at NCBI -

http://

www.ncbi.nlm.nih.gov/tools/primer-blast/It uses Primer3 to design PCR primers and then submits them to BLAST search against user-selected database. The BLAST results are then automatically analyzed to avoid primer pairs that can cause amplification of targets other than the input template.