Building Clone Libraries

Let's have a look at the Materials and Methods of a paper:

"Microbial Community Composition of the Ileum and Cecum of Broiler Chickens as Revealed by Molecular and Culture-Based Techniques"

There's a PDF copy here.

First they describe the animals used, what they were fed, how they were kept, etc. Then there's a bit about culturing some bacteria. They extract and purify their DNA, then go to talk about 16S rDNA Amplification and Cloning. There's a big bit about PCR, so far so good... but then there's this part:

"The products were then purified using a QIAquick PCR purification kit (Qiagen GmbH, Hilden, Germany) and stored at −20°C. The blunt-end PCR products were cloned into linearized pCR-blunt vectors (Invitrogen, Carlsbad, CA), and 1 shot TOP10 competent Escherichia coli cells were transformed using a Zero Blunt PCR cloning kit (Invitrogen) according to the manufacturer’s instructions. Cells were grown on low-salt Luria-Bertani (LB) agar plates (Invitrogen) for 18 to 24 h at 37°C. Colonies were picked randomly and transferred to 1.3 mL SOB-Zeocin medium (Invitrogen) and grown for 24 h at 37°C. Plasmids were purified using a QIAprep 96 Turbo Miniprep kit (Qiagen) using a QIAvac vacuum manifold"

Here's another paper:

"Diversity and Succession of the Intestinal Bacterial Community of the Maturing Broiler Chicken" (PDF)

"The amplified PCR products were purified with the Wizard PCR product purification kit (Promega, Madison, Wis.). The purified products were ligated into pGEM-T Easy (Promega). Ligation was done at 4°C overnight, followed by transformation into competent E. coli JM109 cells by heat shock (45 s at 42°C). The clones were screened for α- complementation of β-galactosidase by using X-Gal (5-bromo-4-chloro-3-indolyl- -D-galactopyranoside) and IPTG (isopropyl- -D-thiogalactopyranoside) (5)." ... "DNA preparations for sequencing were made with the QIAprep spin plasmid kit (Qiagen, Valencia, Calif.) as specified by the manufacturer. Plasmids were eluted with 50 μl of water and stored at 70°C."

What does it mean? One paper's using QIAquick, another one's talking about Wizard. There's some E. coli getting involved there as well... It's confusing. Actually those little paragraphs underlie a lot of lab work.

What is a Clone Library anyway?

Clone libraries are a method of separating rDNA from a PCR sample and creating enough copies to sequence. The basic premise is that rDNA fragments amplified by PCR are inserted into vector DNA (usually plasmids, a loop of DNA commonly found in bacteria), which are then taken up by Escherichia coli. In this context, "competent" means that the bacteria is able to undergo "Transformation" or take up DNA from another source and replicate it. The bacteria are plated out and screened in such a way that each colony on a plate is made up of bacteria carrying a vector with the same bit of rDNA. These can be cultured further to provide enough plasmids to sequence the inserted rDNA or stored for later analysis.

User:Spaully on English wikipedia, Plasmid (english), CC BY-SA 2.5

Purifying DNA from PCR

Although this step is considered optional, it can improve cloning results. At the end of PCR, you've got lots of rDNA, but also lots of other crud from the reactions like primers and enzymes. These and other nonspecific products of PCR can be separated from amplified 16S rDNA using agarose gel electrophoresis (1) or a commercial kits like Wizard or QIAquick⁠. 

Sticky vs Blunt ended PCR products

PCR using Taq polymerase will leave what is known as an A-overhang artefact on amplified DNA. This is an A residue attached to the 3′ end of DNA string. This A-overhang is exploited in some commercial cloning kits to insert amplified rDNA into vector DNA with a complementary T-overhang (TA cloning) (2,3)⁠. This is a "sticky ended PCR product". It should be noted that purification of PCR products using agarose gel electrophoresis removes the A-overhang, so a short step of 3′ adenylation is required after purification (1)⁠.

Vishnu2011, Tacloning, CC BY-SA 3.0

If you haven't used Taq polymerase, you don't have the A-overhang so your PCR products are blunt ended.

Vector DNA

The purpose of vector DNA is to stabilise and replicate an rDNA molecule within a bacterial host. All vector DNA must:

1. Be able to replicate along with the inserted PCR amplicon.
2. Contain unique restriction endonuclease cleavage sites.
3. Contain a marker to distinguish vectors with inserted rDNA, and also distinguish between hosts without vectors.
4. Be relatively easy to extract from the host cell (4)⁠.

The procedure for creating a clone library is outlined in the figure below.

1. Plasmids are mixed with 16S rDNA sequences (iii) and the two spliced together. The insertion point for the 16S rDNA is in the lacZ gene, which codes for the α subunit of β-galactosidase enzyme (ii). This results in insertional inactivation of the lacZ gene (2)⁠. The method for inserting the 16S rDNA into the plasmid will depend on the commercial kit which is being used. The plasmid also contains a gene for antibiotic resistance (i).

2. After insertion of 16S rDNA, the sample contains two kinds of plasmids: Plasmids with the 16S rDNA insertion (i) and plasmids without the 16S rDNA insertion (ii).

3. Escherichia coli are used as host bacteria and are stimulated to take up the plasmids. After this step, there is a mixture of 3 types of E. coli: Those with plasmid type (i), those with plasmid type (ii) and those with no plasmid (iii).

4. The bacteria are then cultured on a media treated with antibiotics to exclude bacteria type iii (2, 3)⁠. The media also contains an inducer for the lacZ gene (so it's expressed) and a substrate for β-galactosidase which turns blue when broken down. Colonies of bacteria which still have an competent lacZ gene due to failure of rDNA insertion into the plasmid will turn blue, allowing for selection of bacteria with a type (i) plasmid (2)⁠.

5. Bacteria from selected colonies are grown overnight in nutrient broth.

So there we have it! Now we've got a theoretically unlimited supply of our 16S rDNA which we extracted from the environmental sample and amplified using PCR. We can now extract the plasmid and inserted DNA using a commercial kit and then sequence it. But wait, as with any technique there are caveats...

Biases of Cloning

There is only one report of a potential bias introduced during the cloning procedure. This focused on comparing the two methods of inserting the 16S rDNA sequences into the plasmid vector, blunt end and sticky end cloning. It was reported that the two methods produced different results when screened using dot-blot hybridisation, however, no phylogenetic details are provided so it is difficult to draw conclusions (5)⁠.

Remember those Heteroduplexes and Chimeras?

Clone libraries may exacerbate the problem of heteroduplex molecules produced during PCR. During cloning in the host bacteria, E. coli DNA repair mechanisms identify the heteroduplex and attempt to repair the mismatched bases. In normal cells, DNA methylation identifies one strand as the correct parent strand. Since neither strand of the inserted DNA is methylated, the repair mechanisms randomly choose one to use a template. For each incorrect base pair, a different strand may be used as the template. The repaired sequences that result are composites of two original strands, referred to as ‘mosaics’ (6)⁠. These are harder to identify than chimeras and so will artificially increase the apparent phylogenetic diversity of a clone library.

Chimeras also present a problem when analysing clone libraries. An analysis of 17 large clone libraries (100 or more clones) of 16S rRNA genes submitted to public databases in 2005 found an average chimera content of 9.0% with one library containing 45.8%. Nine of the libraries had already been checked for chimeras using software (7)⁠. This highlights the importance screening sequences from PCR using reliable chimera hunting software.

How Reliable are Clone Libraries?

As a result of these biases, and those introduced previously by DNA extraction and PCR, it is worth questioning whether clone libraries provide an accurate representation of the qualitative and quantitative composition of microbial communities. Analysis of clone libraries must be viewed objectively and considered as only part of the puzzle of microbial ecology (8)⁠.

Most studies using clone libraries will examine no more than 100 clones, and while this may identify the main taxa present, it is unlikely to represent the true diversity of the orginal sample (8)⁠. While the clone library may not represent true diversity, they benefit from producing longer 16S rDNA fragments for sequencing which provide a greater phylogenetic resolution. Comparing results from different clone libraries is often confounded by the use of different hypervariable regions of 16S rDNA. In light of this, the results of studies using clone libraries should be considered as a semi-quantitative analysis which only superficially explore the true diversity of microbial communities (8)⁠.

References

1. Leigh MB, Taylor L, Neufeld JD. Clone Libraries of Ribosomal RNA Gene Sequences for Characterization of Bacterial and Fungal Communities. Handbook of Hydrocarbon and Lipid Microbiology. 2010. p. 3971–90.

2. Osborn M a, Smith CJ. Molecular Microbial Ecology. Vol. 51. 2009. 370 p.

3. Makkar, H. P S MCS. Methods in Gut Microbial Ecology for ruminants. 2005. 1-223 p.

4. Mullis KB. Recombinant DNA technology and molecular cloning. Sci Am. 1990;Chapter 8:26.

5. Rainey F a., Ward N, Sly LI, Stackebrandt E. Dependence on the taxon composition of clone libraries for PCR amplified, naturally occurring 16S rDNA, on the primer pair and the cloning system used. Experientia. 1994;50(9):796–7.

 

6. Thompson JR, Marcelino L a, Polz MF. Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by “reconditioning PCR”. Nucleic Acids Res. 2002;30(9):2083–8.

7. Ashelford KE, Chuzhanova NA, Fry JC, Jones AJ, Weightman AJ. New screening software shows that most recent large 16S rRNA gene clone libraries contain chimeras. Appl Environ Microbiol. 2006;72(9):5734–41.

 

8. Stackebrandt E, Pukall R, Ulrichs G, Rheims H. Analysis of 16S rDNA clone libraries: part of the big picture. Proc 8th Int Symp Microb Ecol Microb Biosyst new Front Atl Canada Soc Microb Ecol Halifax, Nov Scotia, Canada [Internet]. 1999;1–9