Genetics 466 Topic 3

What reagents are in a PCR reaction?

• DNA template

• Primers

• DNA polymerase

• dNTPs

• Buffer/Cofactors

A number of enzymes involved in DNA replication are not included in a PCR reaction. Why?

A lot of the enzymes are replaced by heat

There is no bubble, the entire strand of DNA is going to be detached via denaturing it

There is only a leading strand, no lagging strand

What are the steps in a PCR reaction?

1. Denature DNA at 95 C

2. Anneal sequence-specific primers of ~20bp at 50-60 C

3. Synthesize new DNA with Taq polymerase at 72 C

4. Repeat the cycle

Why are primers ~20bp long?

We want to make sure that the primer is specific so polymerase knows exactly where to bind

Minimizes off-target replication

Why is annealing temperature typically 50-60 C?

Most of the primer will be able to stick to the DNA at this temperature without going to boiling point

Temperature depends on the primer

Temperature is lower for AT rich and higher for GC rich bonds

Why do we use Taq polymerase for PCR?

This is from a bacteria that lives in boiling temperatures

At the end of the PCR reaction, a DNA fragment of a very specific size is generated. Why?

DNA between the primers is going to be amplified because the primers introduce an end point

Half of the new fragments that are produced in the second round will terminate at the site of the primer introduced in the first round

What is the significance of Sanger dideoxy nucleotide (ddNTP)?

Cannot form a phosphodiester bond with the next incoming dNTP because it has two H at the bottom of the sugar instead of a 3C OH group

How are terminated fragments of many different sizes generated in a DNA sequencing reaction?

Incorporate some dNTPs and ddNTPs to the As (for ex.)

This will lead to a random chain of termination events when an A is incorporated

How do you know the sequence of a specific nucleotide (at a specific position) in a DNA sequencing reaction?

Fluorescence or a gel

How is sequencing with fluorescent chain terminating nucleotides different from the original Sanger dideoxy method?

One reaction is performed with all four ddNTPs. The mixture is run through a capillary that separates fragments by size. A laser detects the color of fluorescence.

This eliminates the need for four lanes on a gel.

Nextgen step 1. Isolate genomic DNA

Isolating many copies of the genome from many cells

Nextgen step 2. Fragment genomic DNA

We can’t read the sequence of long strands of DNA so we fragment it into small ~100 bp parts

The many copies will fragment in different ways

Nextgen step 3. Ligate linkers onto ends of DNA fragments

The ligate linkers are of two varieties to bind to the fragments. Now because they are the same, we can use a primer to attach to these linkers

Nextgen step 4. Bind DNA fragments to chip

Now the fragments are in a static location covalently linked to a chip. Now each spot on the chip represents a different DNA sequence. The strands are going to be amplified via cluster growth. You cannot fluorescently detect each component unless there is multiple to make the signal higher

Nextgen step 5. Denature DNA

Nextgen step 6. Anneal sequencing primer

Nextgen step 7. Add fluorescent, reversible chain terminators

Nextgen step 8. Wash off excess nucleotides and read fluorescence

Nextgen step 9. Cleave off fluorescent chain terminator

Nextgen step 10. repeat cycle from step 7

At the end of the reaction there are many millions of short sequence reads from the fragmented genome

What is a contig?

Computer taking small sequence reads and putting it together in one larger contig

This is basically a chromosome

How are overlapping sequences generated

The computer is searching for the multiple copies of the genome that were fragmented in different ways for overlapping areas based on the reference genome.