Module 4

Nucleotide

Deoxyribose sugar + phosphate group + nitrogenous base

Nucleoside

Deoxyribose sugar + nitrogenous base

Phosphodiester Linkage

Covalent bond between deoxyribose sugar and phosphate

Base Pair Interactions

H bonds holding DNA strands

Between nitrogenous bases

A and T: 2

C and G: 3

DNA Directionality

5’ to 3’

Antiparallel in helix

PCR

Polymerase chain reaction

Create copies of DNA

PCR: dNTPs

Deoxynucleoside triphosphates

Derived from nucleotides

Bind deoxyribose sugar

Release pyrophosphate

PCR: Thermostable DNA Polymerase

Read template DNA

Add dNTPs to 3’ end

Function at higher temp

PCR: Template DNA

Used to determine dNTP addition

PCR: Oligonucleotide Primers

Synthetic single-stranded DNA

1 primer per template strand

Mark amplified section of template

PCR: Cycle Temperatures

1. Melting (>90) to denature DNA double-strand
2. Annealing (50-65) to bind primers to template
3. Extension (68-72) to optimize thermostable DNA polymerase activity

In thermocycler

PCR: Timing

Constant melting and annealing

Variable extension depending on sequence length (amplicon)

PCR: Multiple Cycles

New DNA act as templates

Long initial denaturation

Long final extension

Exponential amplification

High Fidelity Polymerase

Decrease incorrect nucleotide addition

Proofreading activity

Primer Length

Short: Bind other template parts (non-specific)

Long: Self-sticking

Optimal: Tight and specific binding

Primer Melting Temperature

Binding tightness

Proportional to length and GC content

Forward Primer

Same as beginning of forward/template strand

Reverse Primer

Same as end of reverse/complementary strand

Sanger Sequencing Reaction Mixture

Oligonucleotide primer

DNA polymerase

dNTPs

ddNTPs

Template DNA

Sanger Sequencing: Oligonucleotide Primer

Start point for polymerase

Sanger Sequencing: ddNTPs

Dideoxynucleoside triphosphate

Structurally similar to dNTPs

Add to 3’ end

High concentration shorten DNA product

Sanger vs PCR

PCR: 2 primers (double-stranded DNA)

Sanger: 1 primer (single-stranded DNA), ddNTPs

Sanger Sequencing-PAGE Process

1. 4 Sanger sequencing reactions with 1 labeled ddNTP per reaction
2. Separate products by size with PAGE
3. Read labeled nucleotide sequence 5’ (bottom) to 3’ (top)

Dye-Terminator Sequencing Process

1. Sanger sequencing reaction with 4 labelled ddNTPs
2. Capillary electrophoresis separate DNA strands by size
3. Fluorescence detector reveal ddNTP at 3’ end
4. Electropherogram reveal sequence (left to right peaks)

PCR Drawbacks

Exponential amplification

Cannot determine initial DNA amount

No RNA and viral genome detection

qPCR

Quantitative/real-time

Quantify template DNA copies with synthesis rate

qPCR: Cycle Threshold

Low: More template DNA

High: Less template DNA

qPCR: Non-Specific Monitoring

Measure DNA polymerase activity

No info on DNA identity

No amplification differentiation

Intercalators visualize DNA (greater fluorescence over time)

qPCR: Specific Monitoring

Measure DNA amplification

Differentiate specific and non-specific amplification

Oligonucleotide probe bind amplified region (fluorescence relate to DNA concentration)

RT-PCR

Reverse transcription

Detect RNA

RT-PCR Process

1. Reverse transcriptase synthesize single-stranded DNA from RNA template
2. DNA polymerase synthesize complementary cDNA
3. Amplify cDNA (from RNA) to produce double-stranded DNA

RT-qPCR

Reverse transcription qPCR

Quantify RNA

Study gene expression

RT-qPCR Process

1. Reverse transcription of RNA to cDNA
2. Amplify cDNA
3. Measure DNA synthesis (non-specific and specific)

Inducible Transcription

mRNA transcript produced only when molecule/pathway triggered

Antibiotic resistance genes