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’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGCACG 5’ 5’CCGATTACGTCCTAGGCGAACGATGATCGATAGGAAATCGAGGAGGGATCGTAAGGCTGC 3’ 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”

3’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGCACG 5’ CCGATTACGT C C C C C G G G 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. 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)

3’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGCACG 5’ CCGATTACGT C C C C C G G G 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. This single primer is shown in purple ande is the complement of one of the strands of DNA.

3’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGCACG 5’ CCGATTACGT C C C C C G G G 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 before it goes into the thermal cycler! That is one more ingredient has to be added.

3’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGCACG 5’ CCGATTACGT C C C C C G G G G G G A A A A A T T T T T T V V V V V V V A T C G 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’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGCACG 5’ 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’GGCTAATGCAGGATCCGCTTGCTACTAGCTATCCTTTAGCTCCTCCCTAGCATTCGCACG 5’ 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’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.

CCGATTACGTCCTA CCGATTACGTCCT CCGATTACGTCC CCGATTACGTC CCGATTACGTCCTAG CCGATTACGTCCTAGG CCGATTACGTCCTAGGC CCGATTACGTCCTAGGCG CCGATTACGTCCTAGGCGA CCGATTACGTCCTAGGCGAA 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 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.

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. CCGATTACGTCCTA CCGATTACGTCCT CCGATTACGTCC CCGATTACGTC 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. CCGATTACGTCCTA CCGATTACGTCCT CCGATTACGTCC CCGATTACGTC 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. CCGATTACGTCCTA CCGATTACGTCCT CCGATTACGTCC CCGATTACGTC 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. CCGATTACGTCCTA CCGATTACGTCCT CCGATTACGTCC CCGATTACGTC CCGATTACGTCCTAG CCGATTACGTCCTAGG CCGATTACGTCCTAGGC CCGATTACGTCCTAGGCG CCGATTACGTCCTAGGCGA CCGATTACGTCCTAGGCGAA

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 for antibiotic resistance Gene that kills bacteria

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!

Gene for antibiotic resistance Gene that kills bacteria Primer site ON THE VECTOR! Inserted DNA fragment

NEXT STEP-- SEQUENCE THE PRODUCT!

Result!