Transcript Sequencing - University of Vermont

SEQUENCING DNA
Jos. J. Schall
Biology Department
University of Vermont
SEQUENCING DNA
Start with PCR product (your end result of a PCR).
Remember, your “template” DNA in the PCR was extracted DNA
that included thousands and thousands of cells’ worth of DNA,
and you amplified each strand of target DNA about one billion time.
So, you ended up with thousands of billions of copies of the
segment of DNA that was your target.
SEQUENCING DNA
You need to “clean up” your PCR product to get rid of
all the left over primers, taq, template DNA, etc.
You can do this by running the product through a filter, or you
can add enzymes to do the work.
3’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGC
5’CCGATTACGTCCTAGGCGAACGATGATCGATAGGAAATCGAGGAGGGATCGTAAGG
Start with clean PCR product (or could be some other source of pure
piece of the DNA you want to sequence such as a piece that
you had cloned into bacteria, another story). Typically,
it should be under 1500 bases, but ‘reads’ of several kb can
be done. So your new “template DNA” will be the PCR product.
Do another PCR reaction using only one primer that
will anneal at the point indicated in RED.
Therefore, we will be sequencing only the top
strand in the molecule seen above.
This special PCR is called the “sequencing reaction”
C
A
CCGATTACGT
V
CCGATTACGT
G
V
3’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGC
A
T
V G
T
C
T
CCGATTACGT
CCGATTACGT
V
G
T
G
V
V
A
G
A
CCGATTACGT
V
G
C
T
C
T
C
A
The typical PCR mix, except using only ONE primer. This
single primer is shown in purple ande is the complement of
one of the strands of DNA.
HUH?
Doing a PCR with only one primer???
But, the point here is not to do a “real”
Chain reaction, in which the number
Of target strands doubles each cycle,
But each is just increased by one:
NOT: 1,2,4,8,16 (regular PCR)
BUT: 1,2,3,4,5,6 (Sequencing reaction)
C
A
CCGATTACGT
V
CCGATTACGT
G
V
3’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGC
A
T
V G
T
C
T
CCGATTACGT
CCGATTACGT
V
G
T
G
V
V
A
G
A
CCGATTACGT
V
G
C
T
C
T
C
A
The typical PCR mix, except using only ONE primer. This
single primer is shown in purple ande is the complement of
one of the strands of DNA.
C
A
CCGATTACGT
V
CCGATTACGT
G
V
3’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGC
A
T
V G
T
C
T
CCGATTACGT
CCGATTACGT
V
G
T
G
V
V
A
G
A
CCGATTACGT
V
G
C
T
C
T
C
A
Now add something different to your reaction mix before
it goes into the thermal cycler! That is one more ingredient has
to be added.
C
A
CCGATTACGT
V
CCGATTACGT
G
V
3’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGC
A
T
V G
T
C
T
CCGATTACGT
CCGATTACGT
V
G
T
G
A
T
C
G
V
V
A
G
A
CCGATTACGT
V
G
C
T
C
T
C
A
New kinds of bases added! These are ATCG’s
but each one has a colored label, or dye marker
attached. Shown here in four colors. So, you have
“regular” ATCG’s and labeled ATCG’s in the mix.
3’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGCA
CCGATTACGT
We will now follow the sequencing reaction
ith the two kinds of ATCG’s and the single primer.
FIRST: Heat, double strand “melts” and PRIMER ANNEALS
3’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGCA
CCGATTACGTC
First Base is added and by chance it is one of the labeled
bases and the reaction stops there. Why? Because these
fancy labeled ATCG’s have another property. When they
get added to the strand they STOP the reaction on that
single strand.
3’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGCA
CCGATTACGTCC
Or, a normal base is added, then a labeled base.
Reaction stops, but with a longer product.
3’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGCA
CCGATTACGTCCT
Or, two normal bases are added, then a labeled base.
Reaction stops, but with a longer product.
3’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGCA
CCGATTACGTCCTA
Or, three normal bases are added, then a labeled base.
Reaction stops, but with a longer product.
CCGATTACGTC
CCGATTACGTCC
CCGATTACGTCCT
CCGATTACGTCCTA
CCGATTACGTCCTAG
CCGATTACGTCCTAGG
CCGATTACGTCCTAGGC
CCGATTACGTCCTAGGCG
CCGATTACGTCCTAGGCGA
CCGATTACGTCCTAGGCGAA
CCGATTACGTCCTAGGCGAACGATGATCGATAGGAAATCGAGGAGGGATCGTAAG
END RESULT OF SEQUENCING REACTION (A kind of PCR):
Strands of many lengths, from primer + 1 to complete length.
What happened?
During this first cycle, each of the millions of target
strands in the template (your original PCR product)
is copied, sometimes completely, but usually with
1,2,3 etc. fewer bases, always with the last base
With the label.
Then, do this 30 times, and each time the target acts
to make copies again, of lengths:
primer plus one to primer plus all the needed bases.
Next step:
You must “clean up” this product to eliminate
the left over
labeled ATCG’s, primer,
etc. Again, for this you must run it
through a filter that just lets your
labeled product through.
CCGATTACGTC
CCGATTACGTCC
CCGATTACGTCCT
CCGATTACGTCCTA
CCGATTACGTCCTAG
CCGATTACGTCCTAGG
CCGATTACGTCCTAGGC
CCGATTACGTCCTAGGCG
CCGATTACGTCCTAGGCGA
CCGATTACGTCCTAGGCGAA
Then, put this product into the
DNA analyzer instrument. This
instrument has a gel, maybe inside
a capillary tube that looks like a hair
it is so fine. A laser light (yellow) is
at one spot on the gel path. Apply
charge and product starts to move.
CCGATTACGTC
CCGATTACGTCC
CCGATTACGTCCT
CCGATTACGTCCTA
CCGATTACGTCCTAG
CCGATTACGTCCTAGG
CCGATTACGTCCTAGGC
CCGATTACGTCCTAGGCG
CCGATTACGTCCTAGGCGA
CCGATTACGTCCTAGGCGAA
What happens? The molecules move,
but the smallest moves fastest and
reaches the light first, then the next
largest, etc.
CCGATTACGTC
CCGATTACGTCC
CCGATTACGTCCT
CCGATTACGTCCTA
CCGATTACGTCCTAG
CCGATTACGTCCTAGG
CCGATTACGTCCTAGGC
CCGATTACGTCCTAGGCG
CCGATTACGTCCTAGGCGA
CCGATTACGTCCTAGGCGAA
The light records the color of the dye
for each molecule that passes:
Shortest piece = C is seen
Next longest = C is seen
Next longest = T is seen
Next longest = A is seen
Etc.
CCGATTACGTC
CCGATTACGTCC
CCGATTACGTCCT
CCGATTACGTCCTA
CCGATTACGTCCTAG
CCGATTACGTCCTAGG
CCGATTACGTCCTAGGC
CCGATTACGTCCTAGGCG
CCGATTACGTCCTAGGCGA
CCGATTACGTCCTAGGCGAA
The instrument then records the
color of each dye marker it sees
in order. So, the order is….
C, then C, then T, etc.
Read the sequence:
CCTAGGCGAA…..
The instrument is reading off the
sequence of bases.
The instrument then can give
you the results, but also can
show you the “raw” data in the
form a of a special graph, or
“pherogram”.
You can then choose for yourself
to “call” the bases.
Each base will have its own color
on the pherogram see next.
Calling the bases
You can look at the “calls” made by the
instrument, and then can decide to call the
bases yourself. When the instrument calls
an “N” it means it it not sure of the result
and you can look at the graph itself to decide.
Calling the bases
Are you sure of the results? You can
redo the sequencing, but this time with the
other primer, so you can see if the results
are the same (of course, they will be
complements.
This is called sequencing in both directions.
End of story…or actually
the BEGINNING!
The next set of slides shows
how we can get the entire
genome of a species, such as US!
There is not much annotation, so
you are on your own to figure
it out.
SEQUENCING THE HUMAN GENOME
AND
CLONING DNA
GENOMIC DNA
ADD RESTRICTION ENZYMES
Sau3A1
>GATC
CTAG>
Stu1
AGG>CCT
TCC>GGA
DNA DIGESTED INTO PIECES
ADD PLASMID “VECTOR”
IT GRABS UP A PIECE OF DNA
ADD BACTERIA AND PLASMID GOES IN-- ONE
PER BACTERIA CELL
Gene for antibiotic
resistance
PLASMID IN BACTERIA CELL
PLASMID REPRODUCES IN BACTERIA
SPREAD BACTERIA ON PETRI DISH WITH FOOD/AGAR
AND ANTIBIOTIC
EACH BACTERIA CELL SHOWN ENLARGED
NO PLASMID = CELL KILLED BY ANITBIOTIC!
NEVERGROWS!
Gene that kills
bacteria
Gene for antibiotic
resistance
IF NO INSERT, GENE IS ACTIVE AND KILLS CELL!
CELLS NEVER GROW
EACH COLONY IS A CLONE….CONTAINS A PLASMID
WITH ONE PIECE OF DNA
PICK COLONIES, AND PCR EACH ONE
WHAT TO USE FOR PRIMERS??
ON THE VECTOR!
Inserted DNA fragment
Gene that kills
bacteria
Primer site
ON THE VECTOR!
Gene for antibiotic
resistance
NEXT STEP-SEQUENCE THE PRODUCT!
Result!