DNA Replication Technology

PCR Primers Initiate Replication

• Short DNA sequence (oligonucleotides) produced synthetically and added to PCR reaction

Polymerase chain reaction (PCR) cycle

1. Denature (95°C): Split DNA strands - break H-bonds with heat

2. Anneal (prime): Reduce temp, sequence-specific primers bind (anneal) to target DNA (Primers define amplification region “amplicon”)

3. Extend: DNA polymerase extends from primer using target as a template

4. Repeat

What direction does DNA polymerase go?

Adds to 3’ end of primer

Will the primers be part of the final product? Yes

Yes

Primer Design

pick a section of DNA at the 5’ end of each strand, including any sequences you want in the product

Primer Sequence Influences Annealing Temperature

• GC base pairs are stronger than AT pairs, so they can anneal at higher temps

• Longer sequences anneal at higher temps

What is the next nucleotide that DNA Pol will add to the primer (= oligo) sequence?

a. dATP
b. dTTP
c. dGTP
d. dCTP

a. dATP

Goal: design primers to amplify the yellow region of DNA (Insulin gene)

Where should the forward primer bind?

b

Goal: design primers to amplify the yellow region of DNA

What is the sequence of the forward primer?

a.5’TTCATC
b.5’AAGTAG
c.5’TGAGAGG
d.5’ACTCTCC

a.5’TTCATC

Goal: design primers to amplify the yellow region of DNA

Where should the reverse primer bind?

c

Goal: design primers to amplify the yellow region of DNA
What is the sequence of the reverse primer?

b

Why might you want to know a DNA molecule’s nucleotide sequence?

• Find and interpret genes

• Assess evolutionary connections

• Discover and diagnose genetic disorders

Sanger Sequencing

• Similar to PCR, but only one primer is used and no chain reaction occurs

• Dideoxynucleotides (ddNTPs) included to prematurely terminate strand synthesis

Sequencing library prep

• Fragmentation of genomic DNA into small segments

• Adapters added via ligase, sticky end cannot dimerize due to lack of phosphate (DNA needs 3’-OH to 5’P to dimerize)

• PCR amplification based on the known sequence of the adapters

illumina sequencing

• starts with primers attached to a microplate that are complementary to the primers around the DNA to be amplified by PCR

• PCR amplification will make multiple copies of each read that is localized to a specific region of these plates to form identical clusters of sequences

• This allows for a good signal during the actual sequencing step

Illumina nucleotides

• start with a fluorophore attached at the 3’ position (molecule that will shine a specific color in response to focused light)

• This blocks elongation past a single nucleotide until a laser knocks the fluorophore off, which lights up a specific color, letting us know what sequence is added

Shotgun Sequencing

Break many copies of genome into small pieces, use overlaps between pieces from different copies to assemble them properly

ChIP-Sequencing

where specific proteins bind to DNA and where chemical modifications occur on histone proteins across the entire genome

Restriction Enzymes

• bind to dsDNA at specific sequences and cut the DNA, leaving single stranded overhangs.

• If we add a —- recognition sequence to the ends of the PCR product, we will have a much easier time getting the PCR product to stick to the plasmid

• if we can use 2 different —- we can control directionality of insertion

• produces sticky ends and blunt endds

sticky ends

Formed by staggered cuts that leave single-stranded nucleotide overhangs at the 5′ or 3′ end.

Blunt ends

Formed by straight, symmetrical cuts directly across both DNA strands with no single-stranded overhangs

What enzyme will connect the DNA fragments?
a.DNA polymerase
b.Restriction enzyme
c.Ligase
d.Helicase
e.Sliding clamp

c.Ligase