Genomic DNA Library- Preparation and Applications – Genetic Education (2022)

A genomic library is a collection of clones/copies of smaller-sized genomic DNA fragments, used in various downstream applications.

Do you know?

  • The human genome size is ~3.2 billion base pairs.
  • The major part of the genome is non-functional or non-coding (97%).
  • The first human genome sequence was completed in 13 years at the cost of 1 billion USD.

DNA library preparation is an important procedure in high throughput DNA sequencing, microarray and other hybridization-based techniques. Two types of libraries are cDNA libraries and genomic libraries prepared separately for different purposes.

Though the cDNA library-based techniques penetrated deep in genetic science and are popular & valuable; the genomic library, on the other hand, has unique credibility and applications.

Either type of library is a collection of fragments, largely; and are DNA fragments. But the question is why do we need fragments? Why do DNA libraries exist?

Our genome is so large. The unwind DNA strand of all cells can expand up to the sun, so it’s a difficult task to study a single DNA sequence, a part of a gene or a whole gene from the genome.

Technically, scientists have to face many problems, for example, non-complementary base pairing, reaction failure and so on. So the idea is what if we cut off our genome in small size fragments so that we can use a ‘pool of single types of fragments’ to investigate.

Put simply, investigating a 10Kb or 100Kb fragment is way more advantageous than a 3.2Bbp long genome!

Our genome is so huge, much like other eukaryotes where 97% DNA is nonfunctional, only 3% part has functional genes. Besides, It is structurally and functionally complex too. A portion considered as junk previously is now a hot research topic because it regulates gene expression.

Henceforth, evaluating the whole genome is imperative.

To study every possible sequence of our genome, we need it in fragments, accessible fragments, and that’s what our genomic library is. Once we prepare it, we can plan various experiments, determine hypotheses and explore new horizons of our genome.

(Video) genomic DNA library

So what exactly is the genomic library? Why is it needed and how to prepare a genomic library? All these questions and possible others, we will try to answer in this blog.

Stay tuned.

Key Topics:

What is the genomic library?

A genomic library is a fragmented DNA collection of the whole genome of an organism. The fragments are stored in bacterial or yeast vectors later on.

Properties of a genomic library:

  • It should have all the genomic content of the genome of an organism.
  • The size of fragments should be clone-able.
  • The library represents the entire genome.
  • The library should be screenable.
  • Every fragment from the library should be processable enough.

Definition:

A clone/collection of similar types of genomic DNA fragments prepared by techniques like restriction digestion is referred to as a genomic library.

Genomic DNA library preparation:

This type of DNA library preparation process is simple, effective and less complex, unlike the mRNA library preparation. The entire process completes in 5 easy steps:

  • Genomic DNA isolation
  • DNA fragment generation
    • Restriction digestion
    • Mechanical shearing
  • Cloning into a vector system
  • Transforming into a host cell
  • Library screening
Genomic DNA Library- Preparation and Applications – Genetic Education (1)

Genomic DNA isolation:

Genomic DNA isolation has been considered one of the easiest techniques in modern-day genetic science. There are a lot of standard protocols and ready-to-use DNA extraction kits are available on the market.

Keep in mind that the isolated genomic DNA should have purity around ~1.80 (at 260/280 absorbance) and quantity around or more than 100microgram.

You can read our previous article on this topic to understand the basics: Different types of DNA extraction methods.

(Video) Gene Library | Genomic Library and cDNA Library

Put simply, the isolation process includes steps of cell lysis, removal of protein and other impurities, DNA precipitation, DNA purification and DNA elution.

The extracted DNA can be run on 0.8% agarose gel to investigate its purity and quality manually.

DNA fragmentation:

DNA extraction is followed by DNA fragmentation where genomic DNA is parted into smaller random-sized fragments. As aforementioned, two common techniques for the purpose are restriction digestion and mechanical shearing.

But before going further into the article, you can read our previous article on this topic to get more clarity: DNA fragmentation technique.

Restriction digestion:

Restriction digestion is one of the most popular and widely used techniques in genomic library preparation. It’s a process governed by a restriction enzyme that cleaves off DNA into fragments.

The cuts are double-stranded, largely and can produce sticky or blunt ends. The enzyme binds to its recognition site, which is specific, unique and present throughout the genome many times. Take a look at the example below.

Genomic DNA Library- Preparation and Applications – Genetic Education (2)

Sticky ends are more useful and easy to process than blunt ends, henceforth, the only restriction enzyme or endonuclease that can generate sticky ends are selected, for example, EcoR1.

Read more:

  • What is Restriction Digestion and how to do it?
  • What are the Restriction Enzymes? Top 10 Restriction Enzymes used in Genetic Engineering

Mechanical shearing:

A less popular but alternative method for fragmentation is mechanical shearing in which DNA fragmentation is done artificial and generates blunt ends. Mechanical processing needs sub-processing such as adaptor ligation and sticky end generation.

(Video) cDNA library vs Genomic DNA library

Here, a known linker or adaptor DNA is ligated to each DNA fragment and applied for restriction digestion to produce sticky ends.

Cloning into a suitable sized vector:

Now in the next step, processed fragments are incorporated into a suitable vector. In a layman, a vector is a vehicle that transfers the genetic material. Some common vectors used for genomic library preparation are plasmid, cosmid, phage, bacteriophage, lambda phage, BAC or YAC.

Note that vector preparation is an important step for achieving excellent end results. The vector is selected depending upon the size of the genomic DNA fragment. For example, to clone 150 to 200Mb DNA fragments, BAC- bacterial artificial chromosome is the best choice as it can carry 120 to 300 Mb DNA.

Here is the list of vectors, their size and DNA carrying capacity:

VectorSizeDNA carrying capacity in Kb
Plasmid1 to 200Kb15
Phage lambda48.5Kb25
cosmids35-50Kb45
B. Phage35-100Kb70-100
BAC100-200Kb120-300
YAC100-3000Kb250-2000

After completion of the selection process, the vector is processed to incorporate DNA. To do so, the vector DNA which is usually a circular one! Is digestion with the same restriction enzyme as the genomic DNA.

A fragment and vector DNA is ligated using the ligase enzyme and sent for transformation.

Yet another crucial consideration in vector preparation is to use a selectable or screenable marker for validating insert. Screenable markers like the antibiotic gene, incorporated next to the fragment, facilitate the final screening process.

Transformation into host cells:

E.coli and yeast cells are two common organisms used as a host for genomic DNA transformation. The recombinant vector is inserted into the host cell to replicate there. It recombines with the bacterial or yeast chromosome and makes copies of vector genes and fragments of our genomic DNA.

Cells are cultured on agar media containing an antibiotic. So the cells having the antibiotic-resistant gene (our screenable marker) can only grow but not the rest. Meaning, only transformed host cells can grow.

Screening of genomic library:

Largely, two techniques applied for screening genomic fragment inserts are probe-hybridization and polymerase chain reaction. The probe-based hybridization technique is traditional, outdated and less accurate whereas the PCR technique is robust, speedy and more accurate.

In the probe-hybridization-based screening, a known probe sequence of 20 to 200bp, complementary to our genomic DNA fragment is allowed to hybridize and detected using autoradiography.

In the PCR-based amplification technique, a known primer set is allowed to amplify the insert, it also produces clones of that DNA. The untransformed plasmid DNA can’t amplify here.

(Video) Tomasz Dobrzycki: Selecting the right library prep method for your experiment

So the accuracy of PCR is very high and at the same time, it produces additional copies of the genomic fragment too. After completion of the process, the amplicons are separated on agarose gel and stored for further investigations.

This is the simplest representation of how the genomic library is created and maintained. However, it consists of tedious experimental steps like cloning, culturing and transformation.

Advantages of the genomic library:

  • The genomic library preparation process is comparatively simple and easy.
  • A huge number of data can be generated.
  • Fragments can be analyzed to assess SNPs and mutations.
  • It is a comparatively speedy process.
  • The fragment library can be stored for many years.

Disadvantages of the genomic library

  • The size of individual fragments is too large hence sometimes sub-fragmentation is required for some downstream processing.
  • Also, the larger fragment size is difficult to process and treat.
  • The effect of a particular gene or protein can’t be studied.

Applications of Genomic library:

The genomic library preparation technique is widely used for years for DNA sequencing. It is nowadays also used for microarray analysis. Let us understand both scenarios with an example.

Genomic DNA library for DNA sequencing:

Genomic libraries allow scientists to sequence the whole genome using various techniques. Interestingly, the first genome sequenced by Sanger F is achieved by genomic library preparation.

In the process, fragments are re-fragmented by restriction digestion and applied for adapter ligation. Adaptors are known, oligonucleotide sequences, complementary to the primer set used for amplification.

Once every fragment is tagged with adaptors, are simultaneously amplified, sequenced and recorded. Later on, based on the location of the adapter, every sequenced DNA fragment is joined computationally to prepare a sequence of the entire genome.

Genomic DNA library for microarray analysis:

Let’s say we wish to investigate the polygenic nature of 15 different genes and other SNPs. We have all the probes prepared from the data of SNPs of each gene and immobilized on a chip.

Again the genomic DNA is sub-fragmented to increase the resolution of analysis and accuracy of results and are hybridized on a chip. Probes bind if they find complementation and give signals.

Signals are recorded for each probe, are evaluated at the end of the experiment.

Moreover, on a broader note, genomic library preparation has applications in SNP analysis, mutational detection, copy number variation studies and population-based genetic studies.

Wrapping up:

Much like the cDNA library preparation, the genomic library has crucial importance in molecular genetic analysis. In recent times, it has great utility in polygenic disease analysis and investigating thousands of SNPs in a single assay.

However, the process is a bit time-consuming and costlier, it also needed manpower too. Genomes of many different organisms are prepared through the genomic library.

(Video) Building and Screening Genomic Libraries

FAQs

How is genomic DNA library prepared? ›

What are the applications of genomic library? ›

Genomic libraries can be used as substrates to physically map and sequence entire genomes, clone agriculturally important genes and to investigate gene expression patterns. Further, genomic libraries also provide powerful tools and resources for evaluating germplasm conservation stocks and biological diversity.

What is the difference between DNA library and genomic library? ›

The DNA library is a collection of DNA fragments that have been cloned into vectors to recognize and isolate desired DNA fragments.
...
Key Difference between cDNA and Genomic DNA library.
cDNA libraryGenomic DNA library
Size
Smaller compared to genomic DNA libraryVast in comparison to cDNA library
18 more rows

What is a genomic library and what is it used for? ›

Genomic library construction remains an important technique in molecular biology. These resources are critical for analysis of gene function and for detection of related genes from different sources. Genomic libraries are currently in use to find novel natural products, such as antimicrobials.

What are the advantages of genomic library? ›

Genomic libraries offer many advantages, such as being able to study gene regulation, or off target effects of a particular mutation. The large amounts of data allow researchers to better understand how mutations, located outside of the coding region of a gene, affect the organism.

What are two types of gene library? ›

There are basically two kinds of libraries: genomic DNA and cDNA libraries. Genomic DNA libraries contain large fragments of DNA in either bacteriophages or bacterial or P1-derived artificial chromosomes (BACs and. PACs).

What are genomic DNA libraries? ›

A genomic library is a collection of overlapping segments of genomic DNA, cloned into a backbone vector, which statistically includes all regions of the genome of an organism. The resulting cloned DNA is then transformed into a suitable host cell line.

Why is DNA library important? ›

All DNA libraries are collections of DNA fragments that represent a particular biological system of interest. By analyzing the DNA from a particular organism or tissue, researchers can answer a variety of important questions. The two most common uses for these DNA collections are DNA sequencing and gene cloning.

What are the fundamental features of a genomic library? ›

A genomic library contains all the sequences present in the genome of an organism (apart from any sequences, such as telomeres that cannot be readily cloned). It is a collection of cloned, restriction-enzyme-digested DNA fragments containing at least one copy of every DNA sequence in a genome.

Which DNA is restricted to making a genomic library? ›

Which DNA is restricted to making a genomic library? Explanation: Total genomic DNA of an organism is digested using restriction endonuclease and the fragments are inserted into a suitable phage.

What is the difference between DNA and genomic DNA? ›

Genomic DNA and plasmid DNA are two sorts of DNA present in living entities.
...
Difference between Genomic DNA and Plasmid DNA.
Genomic DNAPlasmid DNA
It is a chromosomal DNA larger than the plasmid DNA.It is extrachromosomal DNA that is comparatively smaller.
4 more rows

Which vectors are used for constructing genomic libraries? ›

Bacteriophage lambda vectors are commonly used for construction of genomic libraries. During replication, the phage DNA is produced in a concatameric form, which is cleaved by appropriate endonucleases to allow packaging of a single genome within the phage capsid.

What is stored in genomic library? ›

A genomic library might be a tube full of recombinant bacteriophage. Each phage DNA molecule contains a fragmentary insert of cellular DNA from a foreign organism. The library is made to contain a representation of all of possible fragments of that genome.

What is stored in a DNA library? ›

"DNA libraries" give researchers a way to store and access genes of interest. These libraries consist of large numbers of DNA vectors each containing a different DNA fragment. Different vectors are used for DNA fragments of different sizes.

How do you make a DNA library? ›

To construct a library, total genomic DNA from an organism is cut with one restriction enzyme. The resulting DNA fragments are cloned into a vector, such as ColE1, and then transformed into a suitable host cell. Large numbers of transformants are then kept and screened for the cloned genes.

What is screening of genomic library? ›

Library screening is the process of identification of the clones carrying the gene of interest. Screening relies on a unique property of a clone in a library. The DNA libraries consist of a collection of probably many thousand clones in the form of either plaques or colonies on a plate.

What is genomic library PPT? ›

Genomic library and shotgun sequencing. It includes the topics about genomic library,construction method, its uses and applications, shotgun sequencing, difference between random and whole genome sequencing, its advantages and disadvantages etc.

How can you identify a gene of interest with a DNA library? ›

After cloning all the possible genes from an organism into a library, the next step is to identify the genes of interest. Libraries are often screened by DNA/DNA hybridization using DNA probes. The probes themselves are generally derived from two sources.

How many types of DNA libraries are possible? ›

How many types of DNA libraries are possible? Explanation: There are two types of DNA libraries that can be produced. They are genomic DNA library and cDNA library that is produced from the reverse transcription of the mRNAs produced.

What is genomic library PDF? ›

Collectively known as a genomic 'library', the cloned fragments can be isolated and used for a variety of applications, such as DNA sequencing, production of proteins, analysis of protein interactions, pathway engineering, and searching for related genes in other organisms.

Which method can construct gene library? ›

Phage, bacterial artificial chromosome, yeast artificial chromosome, and other such vectors are suitable for larger DNA. The λ replacement vectors are the most preferred ones for the construction of a genomic DNA library. Usually, the T4 DNA ligase enzyme is used to ligate the chosen DNA sequence into the vector.

What is the difference between genomic DNA and cDNA? ›

The main difference between cDNA and genomic DNA is that cDNA represents the transcriptome of a particular organism whereas genomic DNA represents the genome.

What do you mean by genomic? ›

Genomics is the study of all of a person's genes (the genome), including interactions of those genes with each other and with the person's environment.

What is a recombinant DNA library? ›

A recombinant DNA library typically represents part or all of an organism's genomic DNA or mRNA (represented as cDNA) cloned into vectors and stored as a collection of thousands of transformants.

What are the disadvantages of the genomic library? ›

Disadvantages of DNA libraries: Extraction and sequencing of larger genome is a difficult task. cDNA libraries give a picture of exons only, the alternative splicing os not considered. Contamination of DNA.

Which is used to select genes from genomic library? ›

So, the correct option 'DNA probes'.

What are the limitations of cDNA library? ›

A cDNA library has the drawback that it only includes sequences that are present in mature mRNA. There are no introns and any other sequences that are modified during transcription; sequences that are not transcribed into RNA, such as promoters and enhancers, are also not present in a library of cDNA.

Why genomic DNA is partially digested during genomic library preparation? ›

By carrying out partial restriction enzyme digests with the genomic DNA, they can generate larger fragment sizes. Although it might sound relatively straightforward to use less enzyme and incubate the reaction for less time, partial restriction enzyme digest conditions must be carefully established and monitored.

What is the structure of genomic DNA? ›

Genomic DNA is packaged in histone and non-histone proteins. The DNA is wound around nucleosomes, consisting of two molecules each of H2A/H2B, H3, and H4 plus 145 base pairs of DNA (Figure 13.2C). Between the nucleosomes is “linker” DNA, which is packaged in histone H1.

What is genomic DNA made of? ›

A cell 's DNA, packaged as a double-stranded DNA molecule, is called its genome. In prokaryotes, the genome is composed of a single, double-stranded DNA molecule in the form of a loop or circle; the region in the cell containing this genetic material is called a nucleoid.

What is the size of genomic DNA? ›

The nuclear genome comprises approximately 3 200 000 000 nucleotides of DNA, divided into 24 linear molecules, the shortest 50 000 000 nucleotides in length and the longest 260 000 000 nucleotides, each contained in a different chromosome.

What is gene library write about the types of gene libraries? ›

A gene library is a collection of different DNA sequences from an organism, which has been cloned into a vector for ease of purification, storage and analysis. Gene library is a collection of cloned DNA designed so that there is a high probability of finding any particular piece of the source DNA in the collection.

Why is cDNA used instead of DNA? ›

Advantages of cDNA over Genomic DNA

No introns: Eukaryote genes commonly contain introns (non-coding sequences). These are removed after mRNA synthesis so cDNA contains no introns. This means that a cDNA copy of a gene can be isolated as a single, intron-free fragment.

What is library construction? ›

Fundamental to NGS library construction is the preparation of the nucleic acid target, RNA or DNA, into a form that is compatible with the sequencing system to be used (Figure 1).

What are the basic steps you would need to construct a library containing genomic DNA from a bacterium? ›

Genomic Libraries
  • Isolate a sample of DNA from the organisms cells.
  • Cut the DNA into “gene-size” pieces with a restriction enzyme or enzymes.
  • Cut a cloning plasmid with the same restriction enzymes.
  • Add the cut plasmid and genomic DNA to the same test tube and add DNA ligase.

How are genomic libraries created quizlet? ›

How are genomic libraries created? There are multiple methods - the main one uses molecular cloning to amplify fragments of genomes for further study and addition to a collection (the genomic library).

Which vectors are used for constructing genomic libraries? ›

Bacteriophage lambda vectors are commonly used for construction of genomic libraries. During replication, the phage DNA is produced in a concatameric form, which is cleaved by appropriate endonucleases to allow packaging of a single genome within the phage capsid.

Which DNA is restricted to making a genomic library? ›

Which DNA is restricted to making a genomic library? Explanation: Total genomic DNA of an organism is digested using restriction endonuclease and the fragments are inserted into a suitable phage.

What are the fundamental features of a genomic library? ›

A genomic library contains all the sequences present in the genome of an organism (apart from any sequences, such as telomeres that cannot be readily cloned). It is a collection of cloned, restriction-enzyme-digested DNA fragments containing at least one copy of every DNA sequence in a genome.

How many types of DNA libraries are possible? ›

How many types of DNA libraries are possible? Explanation: There are two types of DNA libraries that can be produced. They are genomic DNA library and cDNA library that is produced from the reverse transcription of the mRNAs produced.

How do you build a DNA library? ›

To construct a library, total genomic DNA from an organism is cut with one restriction enzyme. The resulting DNA fragments are cloned into a vector, such as ColE1, and then transformed into a suitable host cell. Large numbers of transformants are then kept and screened for the cloned genes.

What is the purpose of a genomic library quizlet? ›

A genomic library is a collection of the total genomic DNA from a single organism. The DNA is stored in a population of identical vectors, each containing a different insert of DNA. Vector and Insert. Serves as a carrier for the information encoded by an insert.

What is the difference between a genomic DNA library and a cDNA library prepared for an eukaryote organism quizlet? ›

A genomic library contains fragments of the entire DNA in an organism's genome. A cDNA library contains the coding sequences of eukaryotic genes (minus the introns).

What is a DNA library quizlet? ›

What is a DNA library? a comprehensive collection of cloned DNA fragments from a cell, tissue, or organism.

What are the disadvantages of the genomic library? ›

Disadvantages of DNA libraries: Extraction and sequencing of larger genome is a difficult task. cDNA libraries give a picture of exons only, the alternative splicing os not considered. Contamination of DNA.

Why is genomic library constructed? ›

Genomic libraries derived from eukaryotic organisms are critical for studying the genome sequence of a particular gene of interest. It is useful for prokaryotes with small genomes to identify a clone encoding a specific gene of interest.

What is screening of genomic library? ›

Library screening is the process of identification of the clones carrying the gene of interest. Screening relies on a unique property of a clone in a library. The DNA libraries consist of a collection of probably many thousand clones in the form of either plaques or colonies on a plate.

What is stored in a DNA library? ›

"DNA libraries" give researchers a way to store and access genes of interest. These libraries consist of large numbers of DNA vectors each containing a different DNA fragment. Different vectors are used for DNA fragments of different sizes.

What is genomic DNA? ›

Genomic DNA constitutes the total genetic information of an organism. The genomes of almost all organisms are DNA, the only exceptions being some viruses that have RNA genomes. Genomic DNA molecules are generally large, and in most organisms are organized into DNA–protein complexes called chromosomes.

How can you identify a gene of interest with a DNA library? ›

After cloning all the possible genes from an organism into a library, the next step is to identify the genes of interest. Libraries are often screened by DNA/DNA hybridization using DNA probes. The probes themselves are generally derived from two sources.

Videos

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4. DNA library (genomic and cDNA library) | gene library
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6. Genomic Data Analysis || Introduction for Beginners - Dr. Raghavendran L.
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