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 including AP-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 analysis8) 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

Purine

Pyrim idine

GuanineAdenine

Cytosine Thymine Uracil

Molecular techniques are based on the structure of DNA and RNA

Thymine Adenine

Cytosine

Guanine

Adenine

Guanine

Thymine

Cytosine

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 the gene 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 the 16S-rDNA gene or it can be in a region that is conserved within a specific genus or species such as the nod genes for nitrogen fixation by Rhizobium or the rhl genes for rhamnolipid biosurfactant production by Pseudomonas aeruginosa.

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.

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 to the 3’ end of the primer

denaturation

primer annealing

extension

PCR Round 2

Chromosomal strandLong strand

After the second round of PCR, the number of long strands increases arithmetically and the number of short strands increases exponentially (the number of chromosomal 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

72 0C - primer extension

94 0C - denaturation

Temperature 0C

Temperature control in a PCR thermocycler

94 0C - denaturation

50 – 70 0C - primer annealing

# PCR cycles

[DN

A]

After 25 cycles have 3.4 x 107 times more DNA

plateau is reached after 25-30 cycles

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)

RT-PCRThe enzyme reverse transcriptase is used to make a DNA copy (cDNA) of an RNA template from a virus or from mRNA.

Viral RNA Bacterial mRNA

AAAA3’

Protozoan (eukaryotic) poly A mRNA

PrimerReverse transcriptase

RNA

3’5’

5’

Extension

RNA/cDNA

3’

5’

cDNA

RNA

3’

3’

5’

5 ’

Normal PCR with two primers

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 primer DNA. So performing multiplex PCR can be tricky.

E. Coli genome

Salmonella sp.genome

or

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 of PCR with the downstream and internal primer. A second smaller product is the result of the second round of PCR.

Internal primer

Downstream primer

Upstream primer

ICC-PCR

Integrated cell culture PCR is used for virus detection. Cell culture takes 10 – 15

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

combination of these techniques: grow the sample in cell culture 2 – 3 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.

SYBR Green I

hn

ssD NA -- unbound dyem inimal fluorescence

hn

dsD NA -- bound dye >100fold increase fluorescence

TaqM an -- Hydrolysis P robe

M onitor acceptor fluorescence

hn

H ybridization probes

FRE T

hn

donor acceptor

hn

fluor quencher

hn

E xtension continues

Figure 2. Figure X. Schematic of SYBR Green I, TaqMan, and hybridization probe fluorogenic detection approaches for real-time PCR.

Two labeled probes must bind within the amplicon to generate a fluorescence signal as energy istransferred from the donor fluorophore to the acceptor fluorophore, a process known asfluorescence resonance energy transfer (FRET). These assays can be highly specific since twoprimers and two probes must bind. In addition, detection via hybridization probes is not dependenton a hydrolysis reaction, and thus, a melt analysis can be conducted. These assays can also bemultiplexed.

SYBR Green I is a fluorescent dye that upon intercalation into double stranded DNA exhibits anincrease in fluorescence intensity of greater than 100-fold. Advantages include the relatively lowcost of the dye, the fact that it will work with any primer set since it is not sequence specific, andthe ability to perform a melt analysis. The main disadvantage is that any nonspecific productsformed in the PCR, including primer dimers are also detected.

A TaqMan probe contains a fluorophore in close proximity to a quencher, such that the presence ofthe quencher blocks fluorescence. When bound to the target sequence, the probe is cleaved by thepolymerase. The necessity of a probe to bind internal to the amplicon results in increasedspecificity. These assays can be multiplexed, but have the disadvantages of difficult design andcost of the dual labeled probe and the inability to perform a melt analysis.

Labelling approachesCYBR green

Real-Time PCR

This technique allows quantitation of

DNA and RNA. Reactions are

characterized 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 of cycles. 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

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 Negative microbes

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

RFLP = restriction fragment length polymorphism

RFLP analysis involves cutting DNA into fragments using one or a set of restriction 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

Cloning – the 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 genes

2. Insert genes into an organism, e.g., an overproducer

3. Produce large amounts of a gene

Recombinant DNA techniques

Multiple cloning site

Cloning

CC GGGTCG

C

G

DNA fragment carrying the geneof interest cut w ith restriction enzyme(DNA insert)

Plasm id cut with restrictionenzyme (vector)

CCGG

GT

CG

G

GCC

C

AG

C Recombinant DNA molecule(vector containing DNA insert)

Recombinant DNA

Transformation of bacteriumand selection of a cellcontaining the plasmid

Bacterium

Plasmid

G

TCG

CAG

CBacterium containingthe plasmid

Growth and cell d ivision

Bacteria containingthe clone of the plasmid

F’

G

INA C TIVEC -term inal po lypeptide

INA C TIVEN -term inal po lypeptide

H ost

Vector

H ost+

Vector

H ost+

Vector with DN A insert

INA C TIVEC -term inal po lypeptide

W hite

W hite

Blue

W hite

Selection of recombinants by alpha complementation

Metagenomics

Genetic analysis of an entire microbial community.

Metagenomics involves the cloning of large fragments of DNA extracted from the environment, 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

DGGE – denaturing gradient gel electrophoresis

DGGE is a way to separate multiple PCR products of the same size. These products 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 with some target sequences than others and primers will preferentially amplify targets that are present in high concentration. So scientists still don’t know how accurately this type of analysis depicts the population actually present.

DGGE Analysis

TRFLP Analysis

TRFLP = (terminal restriction fragment length polymorphism analysis)

• A way to separate multiple PCR products of the same size. These products can 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

Automated DNA analyzer

Gel electrophoresis analysis

Fragment Length

0 100 200 300 400 500 600 700

Re

lativ

e A

bu

nd

an

ce

0.00

0.02

0.04

0.06

0.08

0.10

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

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 (green fluorescent protein), beta-galactosidase gene (produces blue color).

insertreportergene

Reporter genes

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.

Affymetrix gene arrays for specific organisms:

Arabidopsis Genome ArraysB. subtilis Genome Array (Antisense) Barley Genome Array C. elegans Genome Array Canine Genome Array Drosophila Genome Arrays E. coli Genome Arrays Human Genome Arrays Mouse Genome Arrays P. aeruginosa Genome Array Plasmodium/Anopheles Genome Array (malaria) Rat Genome Arrays S. aureus Genome Array Soybean Genome Array Vitis vinifera (Grape) Array Xenopus laevis Genome Array Yeast Genome Arrays Zebrafish Genome Array

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:

Phylochip is a microarray with DNA signatures for 9000 known species in the phyla of Bacteria and Archaea to examine “who” is there.

Geochip is a microarray with DNA signatures for various functional genes to examine “what” functions are present