chapter 13 nucleic

8/10/2019 Chapter 13 Nucleic

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Objectives

1) Be able to describe what a gene probe is and what it can be used for.

2) Understand the PCR reaction.

3) Be able to describe the different types of PCR: normal, RT-PCR, ICC-PCR,

multiplex PCR, seminested PCR, PCR fingerprinting, real-time PCR, in situ

PCR. Be able to give an example of the use of each of these types of PCR.

4) Understand the different types of PCR fingerprinting techniques includingAP-PCR, REP-PCR, ERIC-PCR. Be able to give an example application of

a PCR fingerprinting technique.

5) Understand RFLP and its application to forensics.

6) Be able to define cloning, cloning vector, and alpha-complementation.

7) Understand the concept of metagenomic analysis

8) Understand DGGE and TRFLP analysis and its use in community analysis.9) Be able to define what a reporter gene is and know the different types of

reporter genes. Be able to give an example of how each of the different

types reporter genes is used.

10) Be able to define what a microarray is and to give an example of how a

microarray could be used to monitor a microbial community.

Chapter 13 - Molecular Methods


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Purine

Py r im idine

Guani ne

Adenine

Cytosine Thymine Uracil

Molecular techniques are based on the structure of DNA and RNA


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Thym ine Adenine

Cyt osine

Guanine

AdenineGuanine

Thymine

Cytosine


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Gene probes

A gene probe is a short specific sequence of DNA that is used to query

whether a sample contains target DNA, or DNA complementary to thegene probe.

CCTAAAGTGGCATTACCCTTGAGCTA

Single strand of DNA

Target sequence

Gene probe (usually 100-500 bp in length)

ACCGTAAT

The target sequence can be a universally conserved region such as the16S-rDNA gene or it can be in a region that is conserved within a specific

genus or species such as the nodgenes for nitrogen fixation by Rhizobium

or the rhlgenes for rhamnolipid biosurfactant production by Pseudomonas

aeruginosa.


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Need:

Target DNA

Primers: 17 to 30bp, GC content >50%

Primers can be for universal conserved sequences (16S rDNA,dehydrogenase genes) or genus-level conserved sequences (Nod,

Rhl, LamB genes)

dNTPs

DNA polymerase (original was taq polymerase from Thermus

aquaticus. Now there are several other DNA polymerases available)

PCR-Polymerase Chain Reaction

In many cases there is not enough DNA in a sample for a gene probe to

detect. Sample DNA can be amplified using PCR.


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Primer annealing5'

5'3'

3'

target DNA

repeat PCR cycles

5'

5'3'3' Double-stranded DNA

Denaturation5'

5'3'

3'

Extension3'

5'

5'

5'

5'3'

3'

3'

3'

5'

5'

5'

5'3'

3'

3'

Extension

PCR Round 1

DNA polymerase always adds nucleotides tothe 3 end of the primer


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denaturation

primer annealing

extension

PCR Round 2

Chromosomal strand

Long strand

After the second round ofPCR, the number of long

strands increases

arithmetically and the

number of short strands

increases exponentially

(the number ofchromosomal strands is

always the same).

5'

5'

3'

3'

5'

5'3'

3'

3'

5'

5'

5'

5'

3'

3'

3'

3'

5'

5'

5'

5'3'

3'

3'

3' 5'3'5'

5'3'

5'5'3'

3'

3'

5'

5'

3'

Short strand


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72 0C -primer extension

94 0C - denaturation

Temperature 0C

Temperature control in a PCR thermocycler

94 0C- denaturation

5070 0C- primer annealing


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# PCR cycles

After 25 cycles have 3.4 x 107times more DNA

plateau is reached after

25-30 cycles


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A PCR product should be confirmed in at least two ways initially.

These can include:

1. Correct product size.

2. Sequence the product.

3. Use a gene probe to confirm the product.

4. Use seminested PCR (see later)


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RT-PCR

The enzyme reverse transcriptase is used to make a DNA copy (cDNA)

of an RNA template from a virus or from mRNA.

ViralRNA BacterialmRNA

AAAA3

Protozoan(eukaryotic)polyAmRNA

Primer

ReversetranscriptaseRNA

3

5 5

Extension

RNA/cDNA

3

5

cDNA

RNA

3

3

5

5

Normal PCR with two primers


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Multiplex PCR

Use of multiple sets of primers to detect more than one organism or to

detect multiple genes in one organism. Remember, the PCR reaction is

inherently biased depending on the G+C content of the target and primerDNA. So performing multiplex PCR can be tricky.

E. Coligenome

Salmonellasp.genome

or


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Seminested PCR

Three primers are required, the normal upstream and downstream primers as

well as a third, internal primer. Two rounds of PCR are performed, a normal

PCR with the upstream and downstream primer, and then a second round ofPCR with the downstream and internal primer. A second smaller product is

the result of the second round of PCR.

Internal primer

Downstreamprimer

Upstreamprimer


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ICC-PCR

Integrated cell culture PCR is used for virus detection. Cell culture takes 1015

days. PCR alone detects both infectious and noninfectious particles. So use a

combination of these techniques: grow the sample in cell culture 23 days,

release virus from cells and perform PCR. This results in the detection of

infectious virus in a shorter time with a 50% cost savings. It also allows use of

dilute samples which reduces PCR inhibitory substances.


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hn

ssDNA -- unbound dye

minimal fluorescence

hn

dsDNA -- bound dye >100

fold increase fluorescence

TaqMan -- Hydrolysis Probe

M onitor acce ptor fluorescence

hn

Hybridization probes

FRET

hn

donor acceptor

hn

fluor quencher

hn

Extension continues

Labelling approaches

CYBR greenReal-Time PCR

This technique allows quantitation of

DNA and RNA. Reactions arecharacterized by the point in time

during cycling when amplification of a

PCR product is first detected rather

than the amount of PCR product

accumulated after a fixed number ofcycles. The higher the starting copy

number of the nucleic acid target, the

sooner a significant increase in

fluorescence is observed.

TAQ-man probes

FRET probes


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PCR fingerprinting

AP-PCR (arbitrarily primed PCR), 1 primer required, 10-20 bp, no

sequence information required

REP-PCR (repetitive extragenic palindromic sequences) 2 primers

insert randomly into the REP sites

ERIC-PCR (enterobacterial repetitive intergenic consensus sequences),

2 primers insert randomly into the ERIC sites, best for Gram Negativemicrobes

All of these fingerprinting techniques tell one if two isolates are the same

or different. They do not provide information about the identity or

relatedness of the organisms


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RFLP = restriction fragment length polymorphism

RFLP analysis involves cutting DNA into fragments using one or a set ofrestriction enzymes.

For chromosomal DNA the RFLP fragments are separated by gel

electrophoresis, transferred to a membrane, and probed with a gene

probe.

One advantage of this fingerprinting technique is that all bands are bright

(from chromosomal DNA) because they are detected by a gene probe.

AP-PCR, ERIC-PCR, and REP-PCR all have bands of variable

brightness and also can have ghost bands.

For PCR products a simple fragment pattern can be distinguised

immediately on a gel. This is used to confirm the PCR product or to

distinguish between different isolates based on restriction cutting of the

16S-rDNA sequence ribotyping. Also developed into a diversity

measurement technique called TRFLP.

RFLP Fingerprinting Analysis


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Cloningthe process of

introducing a foreign piece

of DNA into a replication

vector and multiplying the

DNA.

Recombinant DNA - foreign

DNA inserted into a vector.

These approaches are used to:

1. Find new or closely related

genes2. Insert genes into an

organism, e.g., an

overproducer

3. Produce large amounts of

a gene

Recombinant DNA techniques

Mu ltip le c lo n in g s ite

Cloning


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F

I N A C T I V E

C - t e r m in a lp o ly p e p t id e

I N A C T I V E

N - t e r m in a lp o ly p e p t id e

H ost

V e ctor

H ost

+

V e ctor

H ost+

V e ctor w i th

D NA i nse rt

I N A C T I V E

C - t e r m in a lp o ly p e p t id e

W h i te

W h i te

B l ue

W h i te

Selection of recombinants by alpha complementation


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Metagenomics

Genetic analysis of an entire microbial community.

Metagenomics involves the cloning of large fragments of DNA extracted from theenvironment, allowing analysis of multiple genes encoded on a continuous piece

of DNA as well as allowing screening of large environmental fragments for

functional activities.

Two main approaches:

sequence analysis of all DNA present

advantage: allows unparalleled access to the genetic information in a sample

disadvantage: difficulty in organization and interpretation of the sequenced

information obtained from complex communities

directed sequencing for identity (16S rRNA gene or a functional gene)

advantage: allows rapid access to specific identity or functional data from an

environmental sample

disadvantage: provides more limited information about the sample


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DGGEdenaturing gradient gel electrophoresis

DGGE is a way to separate multiple PCR products of the same size. Theseproducts can be generated by a 16S-rRNA PCR of community DNA.

DGGE uses either a thermal or a chemical denaturing gradient to separate

bands on the basis of their G+C content.

Once the bands are separated they can be sequenced to allow

identification. The banding patterns themselves can be used to evaluate

whether changes in the population are taking place.

Note of caution: PCR is inherently biased, some primers work better withsome target sequences than others and primers will preferentially amplify

targets that are present in high concentration. So scientists still dont know

how accurately this type of analysis depicts the population actually present.

DGGE Analysis


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TRFLP Analysis

TRFLP = (terminal restriction fragment length polymorphism analysis)

A way to separate multiple PCR products of the same size. These productscan be generated by a 16S-rRNA PCR of community DNA

The PCR is performed as usual with two primers, but one is fluorescently

labeled

The PCR products are then cut up using a restriction enzyme

The fluorescently labeled PCR pieces are detected

TRFLP steps:

1. Extract community DNA

2. Perform 16S rRNA PCR using fluorescently-labeled primer

3. Choose a restriction enzyme for TRFLP that will give the greatest diversity

in restriction product size


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Automated DNA analyzer

Gel

electrophoresis

analysis

Fragment Length

0 100 200 300 400 500 600 700

RelativeAbunda

nce

0.00

0.02

0.04

0.06

0.08

0.10


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Some approaches for analysis of the various bacterial communities

present in environmental samples

1. Culture and identify via 16S-rRNA PCR and sequencing

2. Extract DNA, subject to 16S-rRNA PCR, clone, then sequence

clone libraries

3. Extract DNA, subject to metagenomic analysis

4. Extract DNA, subject to 16S-rRNA PCR, then DGGE analysis

5. Extract DNA, subject to 16S-rRNA PCR, then TRFLP analysis

Discuss the advantages and disadvantages of each of these approaches


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Reporter genes are genetic markers that are inserted into the organism of

interest to allow easy detection of the organism or its activity.

Examples of reporter genes: lux genes (luminescence), gfp genes (greenfluorescent protein), beta-galactosidase gene (produces blue color).

insert

reporter

gene

Reporter genes


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GeneChip microarrays consist of small DNA fragments (referred to also as

probes), chemically synthesized at specific locations on a coated quartz

surface. By extracting, amplifying, and labeling nucleic acids from experimental

samples, and then hybridizing those prepared samples to the array, the amount

of label can be monitored at each feature, enabling either the precise

identification of hundreds of thousands

of target sequence (DNA Analysis) or the

simultaneous relative quantitation of the

tens of thousands of different RNA

transcripts, representing gene activity

(Expression Analysis).

MicroarraysConstructed using probes for a known nucleic acid sequence or for a series of

targets, a nucleic acid sequence whose abundance is being detected.

The intensity and color of each

spot provide information on the

specific gene from the tested

sample.
http://www.gene-chips.com/sample1.jpg


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Affymetrix gene arrays for specific organisms:

Arabidopsis Genome Arrays

B. subtilisGenome Array (Antisense)

Barley Genome Array

C. elegansGenome Array

Canine Genome Array

Drosophila Genome Arrays

E. coliGenome Arrays

Human Genome ArraysMouse Genome Arrays

P. aeruginosaGenome Array

Plasmodium/AnophelesGenome Array (malaria)

Rat Genome Arrays

S. aureusGenome ArraySoybean Genome Array

Vitis vinifera(Grape) Array

Xenopus laevisGenome Array

Yeast Genome Arrays

Zebrafish Genome Array


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Microarray technology is developing fast beyond pure culture:

In 2005, arrays are containing > 250,000 probes.

In 2006, arrays are containings > 500,000 probes.

Microarray analysis is developing the next generation of chips to examine

who is in environmental samples and what they do:

Phylochipis a microarray with DNA signatures for 9000 known species in the

phyla of Bacteria andArchaeato examine who is there.

Geochipis a microarray with DNA signatures for various functional genes to

examine what functions are present

chapter 13 nucleic

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