SEQUENCING DNAJos. J. Schall

Biology DepartmentUniversity of Vermont

SEQUENCING DNAStart with PCR product (your end result of a PCR).

Remember, your “template” DNA in the PCR was extracted DNAthat 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 DNAYou need to “clean up” your PCR product to get rid ofall 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’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGCACG 5’5’CCGATTACGTCCTAGGCGAACGATGATCGATAGGAAATCGAGGAGGGATCGTAAGGCTGC 3’

Start with clean PCR product (or could be some other source of purepiece 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 canbe done. So your new “template DNA” will be the PCR product.

Do another PCR reaction using only one primer thatwill anneal at the point indicated in RED.Therefore, we will be sequencing only the topstrand in the molecule seen above.This special PCR is called the “sequencing reaction”

3’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGCACG 5’

CCGATTACGT

CCGATTACGT

CCGATTACGT

CCGATTACGT

CCGATTACGT

C

C

C

C

C

G

GG

G

G

G

A

A

A

A

A

T

T

T

T

T

T

V

V

V

V

V

V

V

The typical PCR mix, except using only ONE primer. Thissingle primer is shown in purple ande is the complement ofone 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)

3’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGCACG 5’

CCGATTACGT

CCGATTACGT

CCGATTACGT

CCGATTACGT

CCGATTACGT

C

C

C

C

C

G

GG

G

G

G

A

A

A

A

A

T

T

T

T

T

T

V

V

V

V

V

V

V

The typical PCR mix, except using only ONE primer. Thissingle primer is shown in purple ande is the complement ofone of the strands of DNA.

3’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGCACG 5’

CCGATTACGT

CCGATTACGT

CCGATTACGT

CCGATTACGT

CCGATTACGT

C

C

C

C

C

G

GG

G

G

G

A

A

A

A

A

T

T

T

T

T

T

V

V

V

V

V

V

V

Now add something different to your reaction mix beforeit goes into the thermal cycler! That is one more ingredient hasto be added.

3’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGCACG 5’

CCGATTACGT

CCGATTACGT

CCGATTACGT

CCGATTACGT

CCGATTACGT

C

C

C

C

C

G

GG

G

G

G

A

A

A

A

A

T

T

T

T

T

T

V

V

V

V

V

V

V

A

TC

G

New kinds of bases added! These are ATCG’sbut each one has a colored label, or dye markerattached. Shown here in four colors. So, you have“regular” ATCG’s and labeled ATCG’s in the mix.

3’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGCACG 5’CCGATTACGT

We will now follow the sequencing reactionith the two kinds of ATCG’s and the single primer.

FIRST: Heat, double strand “melts” and PRIMER ANNEALS

3’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGCACG 5’CCGATTACGTC

First Base is added and by chance it is one of the labeledbases and the reaction stops there. Why? Because thesefancy labeled ATCG’s have another property. When theyget added to the strand they STOP the reaction on thatsingle strand.

3’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGCACG 5’CCGATTACGTCC

Or, a normal base is added, then a labeled base.Reaction stops, but with a longer product.

3’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGCACG 5’CCGATTACGTCCT

Or, two normal bases are added, then a labeled base.Reaction stops, but with a longer product.

3’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGCACG 5’CCGATTACGTCCTA

Or, three normal bases are added, then a labeled base.Reaction stops, but with a longer product.

CCGATTACGTCCTACCGATTACGTCCTCCGATTACGTCCCCGATTACGTC

CCGATTACGTCCTAGCCGATTACGTCCTAGGCCGATTACGTCCTAGGCCCGATTACGTCCTAGGCGCCGATTACGTCCTAGGCGACCGATTACGTCCTAGGCGAA

CCGATTACGTCCTAGGCGAACGATGATCGATAGGAAATCGAGGAGGGATCGTAAGGCTGC

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 with1,2,3 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 itthrough a filter that just lets

your labeled product through.

Then, put this product into theDNA analyzer instrument. Thisinstrument has a gel, maybe insidea capillary tube that looks like a hairit is so fine. A laser light (yellow) isat one spot on the gel path. Applycharge and product starts to move.

CCGATTACGTCCTACCGATTACGTCCTCCGATTACGTCCCCGATTACGTC

CCGATTACGTCCTAGCCGATTACGTCCTAGGCCGATTACGTCCTAGGCCCGATTACGTCCTAGGCGCCGATTACGTCCTAGGCGACCGATTACGTCCTAGGCGAA

What happens? The molecules move,but the smallest moves fastest andreaches the light first, then the nextlargest, etc.

CCGATTACGTCCTACCGATTACGTCCTCCGATTACGTCCCCGATTACGTC

CCGATTACGTCCTAGCCGATTACGTCCTAGGCCGATTACGTCCTAGGCCCGATTACGTCCTAGGCGCCGATTACGTCCTAGGCGACCGATTACGTCCTAGGCGAA

The light records the color of the dyefor each molecule that passes:Shortest piece = C is seenNext longest = C is seenNext longest = T is seenNext longest = A is seenEtc.

CCGATTACGTCCTACCGATTACGTCCTCCGATTACGTCCCCGATTACGTC

CCGATTACGTCCTAGCCGATTACGTCCTAGGCCGATTACGTCCTAGGCCCGATTACGTCCTAGGCGCCGATTACGTCCTAGGCGACCGATTACGTCCTAGGCGAA

The instrument then records thecolor of each dye marker it seesin order. So, the order is….C, then C, then T, etc.

CCGATTACGTCCTACCGATTACGTCCTCCGATTACGTCCCCGATTACGTC

CCGATTACGTCCTAGCCGATTACGTCCTAGGCCGATTACGTCCTAGGCCCGATTACGTCCTAGGCGCCGATTACGTCCTAGGCGACCGATTACGTCCTAGGCGAA

Read the sequence:CCTAGGCGAA…..

The instrument is reading off thesequence of bases.

The instrument then can giveyou the results, but also canshow you the “raw” data in theform a of a special graph, or“pherogram”.You can then choose for yourselfto “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 theinstrument, and then can decide to call the bases yourself. When the instrument callsan “N” it means it it not sure of the resultand 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 theother primer, so you can see if the resultsare the same (of course, they will becomplements.This is called sequencing in both directions.

End of story…or actually the BEGINNING!

The next set of slides showshow we can get the entiregenome of a species, such as US!There is not much annotation, soyou are on your own to figure it out.

SEQUENCING THE HUMAN GENOME

AND

CLONING DNA

GENOMIC DNA

ADD RESTRICTION ENZYMES

Sau3A1>GATC CTAG>

Stu1AGG>CCTTCC>GGA

DNA DIGESTED INTO PIECES

ADD PLASMID “VECTOR”

IT GRABS UP A PIECE OF DNA

ADD BACTERIA AND PLASMID GOES IN-- ONEPER BACTERIA CELL

Gene for antibioticresistance

PLASMID IN BACTERIA CELL

PLASMID REPRODUCES IN BACTERIA

SPREAD BACTERIA ON PETRI DISH WITH FOOD/AGARAND ANTIBIOTIC

EACH BACTERIA CELL SHOWN ENLARGED

NO PLASMID = CELL KILLED BY ANITBIOTIC!

NEVERGROWS!

Gene for antibioticresistance

Gene that killsbacteria

IF NO INSERT, GENE IS ACTIVE AND KILLS CELL!

CELLS NEVER GROW

EACH COLONY IS A CLONE….CONTAINS A PLASMIDWITH ONE PIECE OF DNA

PICK COLONIES, AND PCR EACH ONE

WHAT TO USE FOR PRIMERS??

ON THE VECTOR!

Gene for antibioticresistance

Gene that killsbacteria

Primer siteON THE VECTOR!

Inserted DNA fragment

NEXT STEP--

SEQUENCE THE PRODUCT!

Result!