Methods used for screening genomic library
December 5th, 2017 | Asim Munawar | No Comments
Genome is the total DNA or total information present on DNA of a certain organism. Human genome has 46 chromosomes or 3 billion base pairs containing intron, axons, functional DNA as well as Junk DNA .The genome in eukaryotes is in form of chromosomes in well-defined nucleus while in prokaryotes the genome is not present in nucleus. For developing a complete genomic library we go on four steps, Isolation of DNA, cleaning, fractionization and cloning. Once a genomic library is prepared, it can be stored and can be helpful in purification, storage and analysis purpose. If we have a DNA fragment and we want to know either this fragment or gene is present in our library or not? If we find it in our genomic library we can re-sequence it analyze.
Keywords: Genomic library, hybridization, RNA, expression, chromosomal walking, DNA
There are four methods used for Library screening.
In this process we need to prepare a probe, probe is a single stranded DNA molecule either labeled with radioactivity or fluorescent protein. Probe is labeled so that it gives fluorescent signals or colored precipitates. Radioactive labeling gives radioactive signals by replacing phosphate backbone with radioactive phosphate (p35).While in non-radioactive probe a specific protein. E.g. biotin is attached because when it reacts with substrate colored precipitates are produced. Probe in some expectations may be RNA.
Next requirement is Nylon membrane and we labeled this membrane so that we can exactly locate required fragment. We paste this membrane on culture plate, bacteria attach to membrane and bacterial lyses occur. Due to lyses bacterial genome is exposed; add certain buffer and process of fixation along with neutralization occur. Double stranded strands are converted into single strands, now expose probe to the membrane and provide the conditions under which probe can hybridize with its complementary fragment. Upon hybridization between probe and its complementary fragment we get signals either fluorescence or coloration. For radioactive probe signal X-ray can be used to clearly indicate signals. Signals tell that where our fragment of interest is present.
We use expression method to find a gene in our library. Vector for cloning is provided with expression system. For screening a gene fragment provide vector with a promoter at 5’ and a terminator at 3’ end. In CDNA library there is always a gene in vector while in genomic library there is a chance to have complete gene in vector, it may be Junk or intron which cannot be transcribed as gene. Our main target is RNA which is product of transcription of gene (DNA). Rest of process is like hybridization. One expression occurs at RNA level and second transcription occurs at protein level. For expression at protein level, promoter is used. Promoter helps to express protein, shift the protein to membrane and provide antibody. Due to antigen-antibody reaction we get signals. This signals indicate protein of require gene in library which also indicates gene of our interest.
- Hybrid arrest and release:
In this method there are two sets of hybridization and transcription. Firstly release hybridization and arrest the transcription. It means hybridization is done and after that transcription is allowed to occur. As in hybridization, bacteria are subjected to lyses, here we will provide enzymes and nucleotides in vitro and there is a gene it will transcribe.
The points in colony where hybridization occurred, the DNA was lyses from double stranded to single strand so at those points there will be no transcription. We are interested in points where no transcription occurred. Now provide certain buffer which will make double stranded DNA into single stranded which remain intact in first process. Again provide in vitro conditions for transcription by arresting hybridization.
- Chromosomal walking:
After target identification we are interested to check what is present in its neighborhood. We need a pair of primers, if a clone fragment is incomplete part of gene, junk or intron, the primers may not attach. But for a gene or complete fragment the primers will attach. As the vector is circular, the primers begin to move in opposite direction and we get two linear strands. After sequencing of these strands, one can get info of sequence at right and left side of fragment of interest.
In DNA libraries, the information is stored as a set of DNA molecules, each of which contains biological sequences that can be used for a variety of applications. 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. The aforementioned genome-wide association studies can identify candidate genes stemming from many functional traits. Genes can be isolated through genomic libraries and used on human cell lines or animal models to further research. Furthermore, creating high-fidelity clones with accurate genome representation and no stability issues would contribute well as intermediates for shotgun sequencing or the study of complete genes in functional analysis.
This article is collectively authored by Asim Munawar1*, Aqsa Arshad1, Muhammad Ishaque Mastoi2, Shehbaz Sharif1 and Muhammad Ali3-1Department of Entomology, University of Agriculture Faisalabad. 2Department of Plant and Environmental Protection, NARC, Park Road Islamabad. 3 Department of Clinical Medicine and Surgery, University of Agriculture Faisalabad.
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