PCR & DNA Sequencing

Polymerase Chain Reaction (PCR)

The amplification of a small amount of DNA into a larger amount

Kary Mullis

Biochemist who developed PCR in 1984

Taq polymerase

DNA polymerase from thermophiles that works at high temperatures (~100°C)

Target DNA

The sample DNA that you want to amplify

Primer

Short DNA strands that bind to target DNA and provide a starting point for replication

dNTPs

Nucleotides used as building blocks for DNA synthesis

PCR contamination

Unwanted DNA from skin cells or bacteria that can interfere with results

Annealing temperature

Temperature at which primers bind to DNA, must be optimized to avoid errors

Magnesium concentration

Affects activity of Taq polymerase

Primer design

Primers must anneal at the same temperature for proper amplification

Too much DNA or polymerase

Leads to nonspecific products and failed PCR

Sanger sequencing

A DNA sequencing method using chain termination (dideoxy method)

ddNTPs

Dideoxynucleotides that terminate DNA chain elongation (lack 3'-OH group)

Purpose of DNA sequencing

Determine gene function and assist in DNA cloning

Sanger sequencing step 1

Split single-stranded DNA into four tubes

Sanger sequencing step 2

Add primer, DNA polymerase, and dNTPs

Sanger sequencing step 3

Add ddNTPs (each tube has one type)

Sanger sequencing step 4

Run samples on polyacrylamide gel

Sanger sequencing step 5

Read DNA sequence from bottom to top of gel

Next-generation sequencing (NGS)

Modern high-throughput DNA sequencing methods

Illumina sequencing

A type of NGS using reversible dye termination

Fragmentation

Breaking DNA into smaller pieces

End repair

Creating blunt ends from fragmented DNA

A-tailing

Adding adenine to 3’ end to allow adapter binding

Adapter ligation

Adding short DNA sequences that serve as primer binding sites

PCR amplification

Amplifying DNA fragments to form clusters

Bridge PCR

Method used to create clonal DNA clusters on flow cell

DNA clusters

Identical copies of DNA fragments for sequencing accuracy

Sequencing by synthesis

Method where nucleotides are added one at a time and detected via fluorescence

Fluorescent nucleotides

Labeled nucleotides detected during sequencing

Flow cell

Surface where DNA fragments attach and are sequenced