Download Sequence - andreawise

DNA and Beyond
2010
PHS
Rashmi Pershad
Objectives
 Learn
about the role of DNA sequencing
and fragment analysis
 Learn how to look up information on any
gene of interest
 How to conduct a BLAST search
 How to go from gene sequence to function
 The $1,000 Genome
DNA Technologies




Methodology
Fragment Analysis
DNA Sequencing
Real time PCR




Applications:
Medical Diagnostics
Forensic Analysis
Plant Genetics
Polymerase Chain Reaction
 PCR
Movie- Cold Spring Harbor
 http://www.dnalc.org/view/15475-The
cycles-of-the-polymerase-chain-reactionPCR-3D-animation-with-no-audio.html
 PCR
Movie Applied Biosystems
 http://media.invitrogen.com.edgesuite.net/
ab/applications-technologies/pharmabiotherapeutics/pcr.swf
Microsatellites
 What
is a microsatellite?
 Are
simple sequence repeats consisting of
1-6 base pair repeats
 How
can they be used?
 They
can be used as genetic markers
Study Microsatellite Instability
377 gels
96 well porous comb gels
Data from 96 lanes in 2.5 hours
Still had to track create size standards and
analyze data.
Fragment Analysis
•Rox 350 size standard 0.02µl per sample
•Fiveplex Microsatellite Analysis
Limitation of Instrumentation
In Cancer Center work with precious archival
paraffin embedded patient DNA. Increased
sensitivity provides more data from limited
sample.
Too much sample results in pull up
Optimal input of product for us is 0.4 ng/µl
DNA Sequencing
Advances in DNA Sequencing
Part I Overview
 Maxam Gilbert Sequencing-(1976)
 Sanger Sequencing
Sequencing has been used to map the human genomes, mutation detection
study genetic diseases such as CF, MS and to study cancer susceptibility genes.
Fluoresent technology
In 1986 ABI developed the technology to sequence using 4 dyes in one lane
fluorescent technology. This allowed for increase in throughput and removed
lane to lane variability.
Platforms
Initially all the work was performed using slab gels.
In the early 1990s 24-36 lane capacity.
In 1996 new instruments allowed for higher throughput 48,64 and 96 lane.
Now capillary electrophoresis
Celera Genomics versus NIH
New technology is whole genome sequencing- Helicos
Dye Primer Labeling
• 4 separate reactions
Taq polymerase
dNTPs
A
A
ACCG A
And ddNTPS
Taq polymerase
dNTPs
C
A
C
ACCG A C
And ddNTPS
G
T
ACC G
ACCGAC T
Dye Terminator labeling
A
Primer
Template
AC
Taq polymerase,
dNTPs
+ ddTerminators
A
C
G
T
Advantages
•Reaction performed in single tube
•Can used unlabeled primers
•False stops are undetected
ACCG
ACCGT
Big Dye Terminators
Argon Laser
Acceptor Dyes
CCD
Camera
dR6G
dRox
dR110
dTamra
linker
Fluorescein
donor Dye
A Emission
C spectra
G
T 500-600nm
Raw
Data
filter
dRhodamine
Acceptor dye
•Donor Dye absorbs excitation energy from laser
•Transfers c.100% to acceptor molecule
•Big Dye gives 2-3 times brighter signal when incorporated into cycle sequencing
product
Protocol
Sequencing
Reagent
DNA
Primer
Big Dye Terminator ready
reaction mix
5X reaction Buffer
deionised water
Total Volume
Reaction Protocol
Quantity
1.5µl
1.6µl
2µl
3µl
11.9µl
20µl
•Set up sequencing reaction in tube.
•Keep reagents on ice
•Mix well and spin briefly
DNA Sequencing Movie
 DNA
Sequencing Movie- Applied
Biosystems
 http://media.invitrogen.com.edgesuite.net/
ab/applications-technologies/pharmabiotherapeutics/DNA_sequencing.swf
DATA FROM 3700 GENETIC ANALYZER RUN
Raw Sequence Data
Start of Sequence
Read out to 800 bases
Standard Template on Pop4 80cm Capillary.
Run under standard conditions
BDv3.1 ( 1µl)- 800-900 base reads
Data from a 3700 in 4hours run
Problematic Template on Pop4 80cm Capillary.
GC rich template with and without Enhancer A
Big dye Version 3.1
Normal Wilm’s Tumor Exon 9
Mutant C
T
What do you do once you have a sequence?
•Compare it to normal sequence.
•Submit it to a sequence alignment program to assemble
sequence
•Search database to identify sequence
DNA Databases





When a scientist sequences a segment of DNA, be it a
single gene, a gene operon or an entire chromosome or
GENOME, the sequence is deposited in an online
database.
The most commonly used database in this country is
NCBI Genbank
Other databases include:
EMBL-EBI (European Molecular Biology LaboratoryEuropean Bioinformatics Institute)
DDBJ (DNA Data Bank of Japan)
These databases contains millions of DNA sequences
and exchange information daily
Bioinformatics Tools
BLAST( NCBI TOOL)
•Sequence Alignment
•Sequence Comparison
•Sequence Identification
Pub Med
 PUBMED
is the NCBI database of
scientific literature that you can search
with terms of interest, to see what
researchers have discovered about the
biology of proteins that are similar to your
query sequence.
BLAST SEARCH

NCBI allows users to search the databases and
perform analyses in various ways.
 You can search by name for nucleotide
sequences (genes) or amino acid sequences
(proteins).
 You can search by name for publications about
the sequence (recorded in the Science Life
literature database called PUBMED).
 You can search for similar sequences using the
feature called BLAST (by inputting all or part of
a DNA or amino acid sequence) and compare
two or more sequences.
NCBI Sequence Record




Each sequence record in the NCBI sequence databases is
organized into three sections:
Header – general information about the sequence including the
organism it came from and the paper in which it was first published.
Features - information about the role of the sequence in the biology
of the organism and any changes that have been made to the
sequence. This section also includes information like the length of
the sequence, the molecular weight of the protein, and any notes
that the depositors wished to add. The start of this section is
indicated with the label FEATURES on the left. Terms beginning
with a / are referred to as ‘qualifiers’. Examples of qualifiers include
/product, /gene, /locus_tag, and /note.
Sequence – nucleotides listed in order and numbered. The start of
this section is indicated with the label ORIGIN on the left.
Reference Material

http://www.pseudomonassyringae.org/Outreach/Module_4_Web.htm
 http://www.digitalworldbiology.com/BLAST
/slide1.html
Questions?
Question 1
 1.
How can you search Genebank for
DNA sequences that are similar to your
gene of interest?
 a) Google
 b) PubMed
 c) MySpace
 d) BLAST
Answer
D
BLAST
Question 2
If your E score is = 0
 a) The sequence is 100% match
 b) The sequences are not alike
 c) There is limited homology between
sequences
 d) Sequences are highly homologous
Answer
A
The e score shows the “expected” match
value. The lower the score the more
significant the alignment .
Question 3
 What
does PCR stand for?
Answer
 Polymerase
Chain Reaction
Exercise 1
You will:
 Copy the sequence provided in the box on the
Blast page of the NCBI :
 websitehttp://www.ncbi.nlm.nih.gov/genome/se
q/BlastGen/BlastGen.cgi?taxid=9606
 Identify the name of the gene,
 Provide the title of the publication where the
sequence was first published, the name of the
journal authors and the full title of the article.
 What did you learn about this gene?
Exercise 1 continued

You will be provided with a list of sequences that
you can use to perform BLAST searches.
 For each sequence provided you will answer
the questions from the previous slide.
 If you need to review a BLAST tutorial you can
find one at:
http://www.digitalworldbiology.com/BLAST/slide1
.html
Sequence 1
TCGAAATAACGCGTGTTCTCAACGCGGTCGCGCAGATGCCTTTGC
TCATC
AGATGCGACCGCAACCACGTCCGCCGCCTTGTTCGCCGTCCC
CGTGCCTC
AACCACCACCACGGTGTCGTCTTCCCCGAACGCGTCCCGGTC
AGCCAGCC
TCCACGCGCCGCGCGCGCGGAGTGCCCATTCGGGCCGCAGC
TGCGACGGT
GCCGCTCAGATTCTGTGTGGCAGGCGCGTGTTGGAGTCTAAA
Sequence 2

GTTTATTAGTGATCATGGCTAAGTTTGCGTCCATCATCGCACTT
CTTTTT
GCTGCTCTTGTTCTTTTTGCTGCTTTCGAAGCACCAACAATGGT
GGAAGC
ACAGAAGTTGTGCGAAAGGCCAAGTGGGACATGGTCAGGAGT
CTGTGGAA
ACAATAACGCATGCAAGAATCAGTGCATTAACCTTGAGAAAGC
ACGACAT
GGATCTTGCAACTATGTCTTCCCAGCTCACAAGTGTATCTGCTA
CTTTCC
TTGTTAATTTATCGCAAACTCTTTGGTGAATAGTTTTTATGTAAT
TTACA
CAAAATAAGTCAGTGTCACTATCCATGAGTGATTTTAAGACATG
TACCAG
ATATGTTATGTTGGTTCGGTTATACAAATAAAGTTTTATTCACCA
Sequence 3

CTCGAGACTAGTTCTCTCTCTCTCTCTCTCGTGCCGCATCTCAC
ACCTGT
GGATGGACGGCAGCTGAACCGCGGGAAACTTTCGTTCTCACTC
TACCTAG
ATGAACTTTAGTTTATATTAAACACGCGTCGACTCCCACACAAA
CCGTGC
TCGTTTTACATCTTTGTCTCCGCTTTTGAAAACGAGAAGTTGAA
TTCGCA
AGACGCAACTTTCCAGCCCCTCACTGAGCGGGCAGAGTCCGT
GAAGCGAT
GGAGCCGTCCGTCATTCCCGGTGCTGACATACCCGACCTTTAC
TCCATTA
ACCCGTTTAATGTCACTTTTCCCGACGACGTTTTGAGTTTCGTT
CCTGAT
GGGAGGAACTACACCGAACCTAACCCGGTAAAGAGCCGCG
GAATCATCA
TCGCCATTTCCATCACCGCTC
Sequence DNA in Thermal Cycler
•Place tubes in thermal cycler
•Repeat the following for 25 cycles
Rapid Thermal Ramp to 96˚C
96˚C for 10 secs
Rapid Thermal ramp to50˚C
50˚C for 5 secs
Rapid thermal ramp to 60˚C
60˚C for 4minutes
•Cool to 4˚C. Hold until ready to purify.