Microbiome Techniques Lecture 8

what are nucleic acid amplification techniques?

Techniques that involve making identical copies of a specific DNA segment in detectable quantities in vitro

nucleic acid amplification techniques are different than cloning in that cloning is

in vivo

nucleic acid amplification techniques exploit _____ DNA replication and ______ mechanisms

in vivo, repair

applications of nucleic acid amplification techniques

Pathogen detection/ quantification (saliva, plaque, blood)

Detection of resistance and virulence genes (if E. coli has particular virulence gene)

Genotyping

Gene expression

Mutations/ allelic discrimination (mainly human, not bacteria)

Preparing libraries for sequencing

methods of nucleic acid amplification techniques

thermal-cycling-dependent, isothermal

thermal-cycling-dependent

Polymerase chain reaction (PCR)

Ligase chain reaction (LCR)

isothermal

Nucleic acid sequence-based amplification (NASBA)

Loop-mediated isothermal amplification (LAMP)

Helicase-dependent amplification (HDA)

Rolling circle amplification (RCA)

Strand displacement amplification (SDA)

not necessary to memorize, just be familiar

"thermal" cycling

raising temperature to split DNA

who invented PCR

Kary Mullis-described in 1987, won nobel in 1993

main three steps of PCR

denaturation, annealing, extension

denaturation

separation of the DNA strands

(94 degrees C)

annealing

Primers hybridize to the separated strands

Temperature depends on stringency required but

generally set at Melting temperature (Tm) minus 5 degrees C

extension

synthesis of the new strands (72 degrees C) by DNA polymerase

how to design primers?

based on whatever sequence we are interested in amplifying that corresponds to GOI

PCR cycle 1

denaturation, annealing, extension occurs

PCR cycle 2

same process of denaturation, annealing, extension occurs for purpose of causing exponential amplification

usually, 30 cycles are done resulting in _________ copies of fragment by the time we are done

1 billion

complete amplification

exponential amplification has occured by end of last cycle

detecting amplicons can be done by

end-point analysis of gel electrophoresis, or fluorescent dye during real-time PCR (qPCR)

types of PCR

conventional or real-time (quantitative-qPCR)

modifications of either type of PCR

Hot-start PCR

Nested PCR

Multiplex PCR

Reverse-transcriptase PCR (RT-PCR)

Random Amplified Polymorphic DNA (RAPD) PCR

RT-PCR

start with RNA, not DNA meant for studying gene expression

essential components of PCR reaction

PCR buffer-where reaction happens

MgCl2 - important for Taq polymerase activity and ensuring specific primer hybridization

dNTP mix

Thermostable Polymerase - at high temperature normal temperature cannot stand it

Sterile deionized, RNAse/DNAse free water

Primer pair(s)

Template DNA/RNA

what is in mastermix

PCR buffer, MgCl2, dNTP mix, thermostable polymerase

optional components of RT- and qPCR reaction

for RT-PCR

Reverse transcriptase

for qPCR

Fluorescent dyes (e.g. SYBR Green)

Probes

commonly used thermostable polymerases

Taq polymerase, Pfu polymerase

Taq polymerase

From Thermus aquaticus

Most commonly used, can tolerate high temperature

Pfu polymerase

From Pyrococcus furiosus

High fidelity applications

High fidelity polymerase

when it synthesizes new strand of DNA, it has a lower rate of error than Taq polymerase because it has a proof-reading mechanism

high fidelity polymerase

DNA extension and misincorporation -> proofreading via exonuclease activity -> DNA extension with correct base

when is high fidelity polymerase used instead of Taq polymerase?

used for sequencing following PCR

primers are _______ nucleotides long

15-25

primer determine ________ of reaction

specificity

design considerations of primers

GC content 40-60% (stable for annealing and hybridization)

GC's evenly distributed

forward and reverse primers Tm with 5 degrees C of each other

Avoid hairpin and primer dimer formation

melting temperature (tm) of primer

how many primers are annealed and how many are not: 4 degrees x G+C + 2 x degrees A+T

10 GC and 10 AT in nucleotide sequence melting temperature

40+20=60 degrees C

primer dimer formation

primers amplify each other

hairpin formation

primers are complementary to each other

SYBR green

real-time detection--non-specific dye that binds to any dsDNA

Taqman probe

real-time detection-hybridizes our target, making sure it is the only thing being amplified and is more specific. Is also more sensitive, paired with special probes at are at both 3' and 5' ends

advantage of a probe over a dye

hybridizes our target, making sure it is the only thing being amplified and is more specific. Is also more sensitive

types of quanitification

absolute and relative

absolute quantification

using digital PCR

Using a standard curve prepared by serial dilution of:

Whole genomic DNA

Plasmid with target sequenced cloned

relative quantification

The quantity of gene in a sample is measured relative

to that in another sample or another gene in the

same sample (calibrator). Two methods can be used:

 Relative Standard Curve Method

 Comparative CT Method (∆∆CT) 3:22

less cycles result in _____ amounts of DNA, and vice versa

higher

Standard curve method

sample is prepared against standard curve 3:20

left=highest amount of DNA (fewer cycles)

right=least amount of DNA (more cycles)

hot-start PCR

bind antibodies to DNA polymerase--when temperature is raised, PCR is activated. Prevents non-specific amplification during the set up of PCR reaction

nested PCR

makes reaction more specific and more sensitive

two cycles, first targeting a larger fragment, second uses nested primers to amplify again

multiplex PCR

instead of doing separate reactions for multiple samples, do everything in one tube. use different probes to label each thing you are targeting (ex: blue for HIV, green for HPV, etc). cannot use SYBR green because it amplifies any dsDNA in the sample

Reverse Transcriptase PCR

mRNA converted into complementary DNA using reverse transcriptase enzyme. we then do PCR with cDNA. can be done in one tube (everything done at once) or two tubes (RT added first, then PCR done in another tube)

why would you do RT-PCR in two tubes instead of one?

if you want to do multiple reactions. RT added to small amount of RNA, then a lot of cDNA is made

common problems with PCR

false positive, false negative

preventing false positive in PCR

Avoid contamination:

Separate working areas (extraction room - clean room, post-PCR room, etc)

Using laminar flow/PCR cabinets with UV light

Aliquoting of reagents

Including a negative control (from the extraction step). should not amplify

Real-time PCR instead of conventional PCR (closed-tube. conventional PCR involves opening tube which risks contamination)

Use of uracil-DNA glycosylase (UDG)

Use barrier (filter) disposible dip

Autoclave tools and instruments

Avoid non-specific amplification

principle of eliminating contaminant using UDG

breaks up Uracil from contaminant-DNA that may have carried over from previous reaction

principle of preventing contaminants using filter tips

when pipetting, no DNA goes into tip (like a mask)

principle of avoiding non-specific amplication

Design specific primers and probes

Avoid formation of primer-dimers

Use stringent reaction conditions (temp., MgCl2 concentration, etc)

Use HOTSTART taq polymerase! (so no reactions occur that could cause false positives)

preventing false negatives in PCR

Use sensitive nucleic acid extraction methods (make sure you extracted good amount of DNA/ RNA from sample)

Control for PCR reaction inhibitors - include internal control, which means spiking sample with non-sequenced DNA (reference genes)

Appropriate reaction conditions - include positive control! (ex: if testing for HIV in a sample, test your PCR on a sample that you KNOW has HIV)

Make sure primers cover all genotypes/ subtypes

Avoid nucleic acid degradation

to account for false positives, have _____ controls; to account for false negative, have _____ controls

positive (disease you are testing for), negative (water)

Avoid nucleic acid degradation

Store at appropriate temp. (4 degrees C; -80 degrees C)

Using DNAse and RNAse tips and tubes can cause DNA/ RNA to be broken down