DNA Libraries / Genomic DNA vs cDNA .pdf

This pdf is about the DNA Libraries / Genomic DNA vs cDNA.

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1. selfexplanatory.2022
HelloHI
नमस्ते
ْم
ُ
كْيالاع ُمالََّسلا
َِّللَّٱ ُةامْحاراو
ُهُتاكاراباو
Saba Parvin Haque
M.Sc. Life Sciences
(Specialization in Neurobiology)
from “Sophia College”
(Autonomous), Mumbai.

2. DNA
libraries
https://images.app.goo.gl/AQRHJ5dnrVZgLkde9
https://images.app.goo.gl/eTvyU23f8TxnzHG6A

3. INTRODUCTION
•Gene sequence are arranged in genome in a random fashion and selecting or isolating a
gene is a big task especially when the genomic sequences are not known.
•A small portion of genome is transcribed to give mRNA where as a major portion
remained un-transcribed.
•Hence, there are two ways to represent a genomic sequence information into the multiple
small fragments in the form of a library:
•(1) Genomic library (2) cDNA library.
https://images.app.goo.gl/YhwLB5j9MnGzgsLT7

4. PREPARATION OF GENOMIC LIBRARY
•A genomic library represents complete genome in multiple clones containing small DNA fragments.
•Depending upon organism and size of genome, this library is either prepared in a bacterial vector or in yeast
artificial chromosome (YAC).
•Construction of genomic library is given in it has following steps:
1.Isolation of genomic DNA
2.Generation of suitable size DNA fragments
3.Cloning in suitable vector system (depending on size)
4.Transformation in suitable host.

5. 1. ISOLATION OF
GENOMIC DNA
https://nptel.ac.in › mod3PDFLecture 10: DNA Libraries (PART-I) - NPTEL

6. 1. ISOLATION OF GENOMIC DNA HAS FOLLOWING STEPS:
1. Lysis of cells with detergent containing lysis buffer.
2. Incubation of cells with digestion buffer containing protease-K, SDS to release genomic DNA from DNA-
protein complex.
3. Isolation of genomic DNA by absolute alcohol precipitation.
4. Purification of genomic DNA with phenol: chloroform mixture. Chloroform: phenol mixture has two phases,
aqueous phase and organic phase. In this step, phenol denatures the remaining proteins and keep the protein in
the organic phase.
5. Genomic DNA present in aqueous phase is again precipitated with absolute alcohol.
6. Genomic DNA is analyzed on 0.8% agarose gel and a good preparation of genomic DNA give an intact band
with no visible smear.

7. 2. GENERATION OF SUITABLE SIZE FRAGMENTS
•Restriction digestion: Genomic DNA can be digested with a frequent DNA cutting enzyme such
as EcoR-I, BamH-I or Sau3A to generate the random sizes of DNA fragments.
•The criteria to choose the restriction enzyme or pair of enzymes in such a way so that a reasonable
size DNA fragment will be generated.
•As fragments are randomly generated and are relatively big enough, it is likely that each and
every genomic sequence is presented in the pool. As size of the DNA fragment is large, complete
genome will be presented in very few number of clones. In addition, genomic DNA can be
fragmented using a mechanical shearing.

8. •If a organism has a genome size of 2x107 kb and an average size of the fragment is 20kb, then no. of
fragment, n= 106.
•In reality, this is the minimum number to represent a given fragment in the library where as the actual number
is much larger.
•The probability (P) of finding a particular genomic sequence in a random library of N independent clone is as
follows:
•N=ln (1-P)/ln (1-f)
•Where, N=number of clones, P=probability, n= size of average fragment size.
2. GENERATION OF SUITABLE SIZE FRAGMENTS

9. HOW TO SELECT A VECTOR ?
where,
Nis the necessary number of recombinants.
P is the desired probability that any fragment in the genome will occur at
least once in the library created.
F is the fractional proportion of the genome in a single recombinant
fcan be further shown to be:
f=i
g
Where,
iis the insert size
gis the genome size.

10. VECTOR SELECTION EXAMPLE
•The above formula can be used to determine the 99% confidence
level that all sequences in a genome are represented by using a vector
with an insert size of twenty thousand basepairs (such as the phage
lambda vector). The genome size of the organism is three billion
basepairs in this example.
•Thus, approximately 688,060 clones are required to ensure a 99%
probability that a given DNA sequence from this three billion
basepairs genome will be present in a library using a vector with an
insert size of twenty thousand basepairs.

11. 3. CLONING INTO THE SUITABLE VECTOR
•The suitable vector to prepare the genomic library can be selected based on size of the fragment of genomic
DNA and carrying capacity of the vector.
•Size of average fragment can be calculated and accordingly a suitable vector can be chosen.
•In the case of fragment generated by restriction enzyme, vector can be digested with the same enzyme and
put for ligation to get clone.
•In one of the approach's, a adopter molecule can be used to generate sticky ends, alternatively a
endonuclease can be used to generate sticky ends.

12. 4. TRANSFORMATION TO GET COLONIES
•Post ligation, clones are transformed in
a suitable host to get colonies.
•A suitable host can be a bacterial strain
or yeast.
https://nptel.ac.in › mod3PDFLecture 10: DNA Libraries (PART-I) - NPTEL

13. CONSTRUCTION OF cDNA LIBRARY
•A cDNA library represents mRNA population present at a particular stage in a organism into multiple clones
containing small DNA fragments.
1. Isolation of mRNA
2. Preparation of complementary DNA fragments
3. Cloning in suitable vector system
4. Transformation in suitable host.

14. ISOLATION OF mRNA (MESSENGER RNA)
•It has a CAP structure at 5‟, coding sequence and a poly A tail at its 3‟ region.
•The Nucleotide A forms 2 hydrogen bonding with nucleotide T and this pairing is very specific.
•Exploiting this feature, m-RNA population can be isolated from RNA pool using a poly-T affinity column.
https://nptel.ac.in › mod3PDFLecture 10: DNA Libraries (PART-I) - NPTEL

15. The Steps in m-RNA isolation from cell is given below.
1. Release of total RNA either by a lysis buffer containing detergent or by homogenization in the case of hard
tissue.
2. Mixing of poly-T containing beads with the total RNA preparation. Due to mutual exclusive affinity,
mRNA binds to the poly-T beads.
3. Wash the beads with washing buffer to remove non-specific cross contaminating species.
4. Elute the mRNA from beads; its purity can be checked on polyacrylamide gel.
ISOLATION OF mRNA (MESSENGER RNA)

16. https://nptel.ac.in › mod3PDFLecture 10: DNA Libraries (PART-I) - NPTEL

17. PREPARATION OF COMPLEMENTARY DNA ( cDNA)
•Multiple approaches have been developed to prepare complementary DNA (cDNA) from isolated
mRNA. In all approaches the 3 steps are performed.
1. First strand synthesis with a reverse transcriptase
2. Removal of RNA template
3. Second strand synthesis.

18. HOMOPOLYMER TAILING
•This method exploits the presence of poly A tail present on m- RNA to synthesize first DNA strand followed by
degradation of RNA template and synthesis of second strand. It has following steps:
1. An oligo dT primer is used with mRNA as template to prepare first strand of DNA with the help of reverse
transcriptase and dNTPs.
2. After the synthesis of first strand, terminal transferase is used to add C nucleotides on 3‟of both mRNA and newly
synthesized first strand of DNA.
3. DNA: RNA hybrid is loaded on a alkaline sucrose gradient. This step will hydrolyze RNA and allow the full
recovery of cDNA.
4. Next, an oligo dG primer is used with cDNA as template to prepare second strand of DNA with the help of reverse
transcriptase and dNTPs.

19. https://nptel.ac.in › mod3PDFLecture 10: DNA Libraries (PART-I) - NPTEL

20. GUBBER-HOFFMAN METHOD
•The method of Gubber-Hoffman.
•In this approach, after first strand synthesis using oligo dT primer in the presence of reverse transcriptase
and dNTPs.
•DNA:RNA hybrid is treated with Rnase H to produce nicks at multiple sites.
•Then DNA polymerase is used to perform DNA synthesis using multiple fragment of RNA as primer to
synthesize new DNA strand.
•This method produces blunt end duplex DNA product.

21. https://nptel.ac.in › mod3PDFLecture 10: DNA Libraries (PART-I) - NPTEL

22. CLONING OF cDNA INTO THE VECTOR
•The cDNA is ligated into the suitable vector to generate clone.
•Transformation to get colonies
•Post ligation, clones are transformed in a suitable host to get colonies. A suitable host can be bacterial
strain or yeast.

23. Figure: Steps in construction of cDNA library.
https://nptel.ac.in › mod3PDFLecture 10: DNA Libraries (PART-I) - NPTEL

24. GENOMIC DNA VS cDNA

25. APPLICATIONS OF GENOMIC LIBRARY
1.Genomic library construction is the first step in any DNA sequencing projects.
2.Genomic library helps in identification of the novel pharmaceutically important genes.
3.Genomic library helps in identification of new genes which were silent in the host.
4.It helps us in understanding the complexity of genomes.
5.Serving as a source of genomic sequence for generations of transgenic animals through genetic
engineering.
6.Study of the function of regulatory sequences in vitro.
7.Create cDNA libraries to determine what genes are being expressed at a particular time.

26. APPLICATIONS OF cDNA
1.Storage of reduced amount of information due to the
removal of non-coding regions.
2.cDNA can be directly expressed in prokaryotic organisms.
3.cDNA libraries are useful in reverse genetics where the
additional genomic information is of less use.
4.cDNA library is useful for isolating gene that codes for
particular mRNA.
5.cDNA library is a powerful and useful tool in the area of
biotechnology.
6.It is helpful in expressing eukaryotic gene in prokaryotes,
which helps in the transcription process of prokaryotes.
7.It is used to isolate DNA sequences to code mRNA.
8.Advantage of cDNA library is to isolate homologous genes.
9.It is also used for the screening genomic libraries to isolate
specific cDNA.
10.cDNA of protein can facilitate to generate antibodies and
monoclonal antibodies.
11.The most important application of cDNA library is to study
expression of mRNA.

27. REFERENCES
•Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2007). Molecular Biology of the Cell, 5th Edition (5th
ed.). Garland Science.
•Ma, Hongbao. (2016). Gene Library. 9. 19-23. 10.7537/marsnys091016.04.
•https://pediaa.com/difference-between-cdna-and-genomic-dna/

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