PCR and DNA sequencing

Polymerase chain reaction (PCR)

Polymerase chain reaction (PCR)

a technique for amplifying DNA in vitro by incubating it with specific primers, a heat-resistant DNA polymerase, and nucleotides

materials for PCR

A thermophilic DNA polymerase 2 primers, which anneal to opposite strands 5' dNTPs Salts, buffers etc. to keep the protein happy

how do PCR primers work

PCR needs 2 primers: forward and reverse, these must anneal to opposite strands and should be oriented such that the extension from 5' to 3' synthesize DNA towards the sequence that corresponds to the other primer

Denaturing PCR

DNA "melting" at high temperature (95 degrees C) to break hydrogen bonds that hold strands together

Annealing PCR

binding of primers (55 degrees C), instead of DNA strands coming back together, primers bind to two strands

Extension PCR

once the primers are annealed to DNA, DNA polymerase can extend them by adding new nucleotides

Number of perfect fragments after n cycles of PCR

2^n-2n

why do we use thermophilic DNA polymerase in PCR

since the DNA is heated at the beginning of every cycle of PCR, it is easier to use a DNA polymerase that works most efficiently at higher temperatures than a regular DNA polymerase that would denature after every cycle and have to be replaced

Real time PCR (qPCR)

Add ethidium bromide (EtBr) to to tube, which is only fluorescent when bound to DNA If PCR works, we do not need a gel, we can measure the fluorescence in the tube instead of running a gel, we will get the same answer quickly and quantitatively Measure fluorescence in real time, using a fancier PCR machine

Gel electrophoresis

a technique for separating nucleic acids or proteins on the bases of their size and electrical charge, both of which affect their rate of movement through an electric field in a gel made of agarose or another polymer

Polyacrylamide gel electrophoresis (PAGE)

used to look at smaller pieces of DNA/RNA at a higher resolution Can resolve DNA molecules whose length differ by 1 bp, also used to separate proteins

what is RT-PCR

(reverse transcription PCR) is used to amplify RNA cDNA copy is made by reverse transcription, then amplified by PCR

how does RT-PCR work?

Prime reverse transcription using reverse primer Synthesize cDNA using reverse transcriptase PCR. First cycle generates double stranded DNA, Second cycle similar to the first cycle of a normal PCR reaction

cDNA

complementary DNA, DNA synthesized from a single-stranded RNA template in a reaction catalyzed by reverse transcriptase

what is sangar sequencing?

copy the DNA in vitro (i.e. in a tube) with large amounts of dNTPs and small amounts of ddNTPs ddNTPs terminate DNA synthesis at a given nucleotide (A,T,C,G) when enzyme adds it instead of dNTP If we do equivalent reactions with ddCTP, ddGTP, and ddTTP, we get a series of bands in each lane that allows us to read the sequence If we label ddNTPs in unique colors, length of DNA molecule tells us the position in the DNA strand; the color tells us what nucleotide was added We can "walk" along the DNA, designing new primers as we go

ddNTP

has a hydrogen rather than a hydroxyl group at the 3' carbon so it cannot be extended by a DNA polymerase, a chain terminator

Next generation sequencing process (NGS)

Fragment DNA in small pieces add DNA adaptors (ensure all DNA molecules start and end with the same sequence as the other molecules), allows us to amplify everything by PCR using same primers Dilute and immobilize DNA beads or in microwells, one molecule per bead/well Then, amplify so the bead/well contains many copies of the identical DNA molecules (i.e. clonal DNA) Add one dNTP at a time to wells, when it is the correct dNTP, the release of the phosphate group will fluoresce Use microscope to catch and count fluorescence events on millions of beads Computer translates fluorescence events into DNA sequence

what happens if multiple consecutive nucleotides are added in NGS?

the flash of light is brighter- proportional to the number of nucleotides