Germplasm or genetic resources are stored in the form of seeds in a specialized place known as gene bank.

 

Gene banks are a type of bio repository which preserve genetic material.

For plants this is done by in-vitro storage, freezing cuttings from the plants or stocking the seeds.

PROCEDURE OF MAKING cDNA LIBRARY-

A cDNA library is a combination of clone cDNA (complimentary DNA) fragments inserted into a collection of host cells, of which constitute some portion of the transcripton of the organism and are stored as a library.

cDNA is produced from fully transcribed mRNA found in the nucleus and therefore, contains only the expressed genes of an organism.

A cDNA library represents mRNA population present at a particular stage in an organism into multiple clones containing small DNA fragments. It has following steps:

o
• Isolation of mRNA – The structure of typical mRNA is given below:

It has a CAP structure at 5’ , coding sequence and a polyA tail at its 3’ region.

The nucleotide A forms 2 hydrogen bonding with nucleotide T and this pairing is very specific. Exploiting this feature, mRNA population can be isolated from RNA pool, using poly-T affinity column.

The steps in mRNA isolation from cell is given below:

o
• Release of total RNA either by a lysis buffer containing detergent.
o
• Mixing of poly-T containing beads with the total RNA preparation. Due to mutual exclusive affinity, mRNA binds to the poly-T beads.
o
• Wash the beads with washing buffer to remove non specific, cross contaminating species.
o
• In new to the mRNA from beads, its purity can be checked from poly acrylamide gel.

 

o
• Preparation of complementary DNA- Multiple approaches have been developed to prepare complementary DNA (cDNA) from isolated  mRNA. In all approaches the three steps are perfomed.

o
• First strand synthesis with a reverse transcriptase.
o
• Removal of RNA template.
o
• Second strand synthesis.

 

         Homopolymer tailing:-

This method exploits the presence of poly-A tail present on mRNA to synthesize first DNA strand followed by degradation of RNA template and synthesis of second strand. It has following steps:

o
1. An oligo dT primer is used with mRNA as a template to prepare first strand of  DNA with the help of reverse transcriptase and dNTPs.
o
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.
o
3. DNA:RNA hybrid is loaded on an alkaline sucrose gradient. This step will hydrolyze RNA and allow the full recovery of cDNA.
o
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.

o
• Cloning of cDNA into the vector- The cDNA is ligated into the suitable vector to generate clone.
o
• 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.

 

 

ADVANTAGES OF cDNA LIBRARIES-

o
• Ease of studying the genome of different organisms.
o
• Identification of genes in an organism.
o
• Creation of transgenic organism.
o
• Gene therapy.
o
• Pharmaceutical Applications through the study of genetic defects.

 

 

 

CONSTRUCTION OF GENOMIC LIBRARY-

Gene sequences are arranged in genome in a random fashion and selecting or isolating is a big task especially when the genomic sequences are not known.

A small portion of genome is transcribed to give mRNA where a major portion  remains untranscribed.

PREPARATION OF GENOMIC LIBRARY-

A genomic library represents complete genome in multiple clones, containing small DNA fragments. Depending upon the organism and size genome, this library is either prepared in a bacterial vector or in a yeast artificial chromosome (YAC). It has following steps:

o
• Isolation of genomic DNA- Isolation of genomic DNA has following steps:

o
• Lysis of cells with detergent containing lysis buffer.
o
• Incubation of cells with digestion buffer containing protease-

K, SDS to release genomic DNA from DNA-protein complex.

o
• Isolation of genomic DNA by absolute alcohol precipitation.

o
• Purification of genomic DNA with phenol : chloroform  mixture. In this step, phenol denatures the remaining proteins and keep the protein in oragnic phase.
o
• Genomic DNA present in aq. phase  is again precipitated with absolute alcohol.
o
• Genomic DNA is analyzed on 0.8% agarose gel and a good preparation of genomic DNA give an intact band with no visible smear.

 

o
• Generation of suitable size fragments- Next step generation of genomic DNA into suitable size fragments.

             Restriction digestion-

    Genomic DNA can be digested with a fragment DNA cutting enzymes such as EcoR-I or BamH-I to generate the random sizes of DNA fragments. As fragments are randomly generated and are relatively big enough, it is likely that each and every genomic sequence is presented in a 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. The probability (P) of finding a particular genomic sequence in a random library of  N  independent clone is as follows:

         N = In (1-P)/ In (1-1/n)........ (Eq. 10.1)

o
• 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 of the vector (Table 10.1).

                    Size of average fragment can be calculated from the Eq. 10.1 and accordingly a suitable vector can be chosen.

o
• In the case of fragment generated by restriction enzyme, vector can be digested with the same enzyme and put for ligation to get clone.
o
• In the case of mechanical shearing mediated fragment generation putting these fragment needs additional effort.
o
• In one of the approaches, a adopter molecule can be used to generate sticky ends, alternatively a endonuclease can be used to generate sticky ends.

o
• 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.

 

METHODS OF SCREENING cDNA & GENOMIC LIBRARIES-

In a typical experiment, host bacteria containing either a plasmid-based or bacteriophage- based library are plated out on a petri-dish and allowed to grow overnight to form colonies.

         A replica of a bacterial colonies is then obtained by overlaying the plate with a nitrocellulose disc. The disc is removed, treated with alkali to dissociate bound DNA duplexes into single-stranded DNA, dried, and placed in a sealed bag with labelled probe. If the probe DNA is duplex DNA, it must be denatured by heating at 70C.

The probe & the target DNA complementary sequences must be in a single stranded form if they are to hybridize with one another. Any DNA sequences complementary to the probe DNA will be revealed by autoradiography of nitrocellulose disc.

                                Bacterial colonies containing clones bearing target DNA are identified on the film and can be recovered from master plate.

 

SCREENING BY DNA HYBRIDIZATION-

DNA sequence information can be exploited with a general rule that nucleotide present in a DNA sequence provides a specificity due to unique base pairing preferance of nucleotides.

                             A=T                         G=C

As a result a particular DNA sequence can be identified by a complementary single stranded sequence.

The DNA sequence used for this purpose is called ‘Probe’. The position of probe is the actual site of desirable clone of containing specific sequence. It has following steps:

o
• Preparation of radioactive probe- There are two different method used to label a single stranded DNA probe either at terminal or through out the sequence.

o
1. Random primer method- In this method, a random primer is used to anneal to the template and then a PCR reaction is performed in the presence of radio labeled nucleotide. After PCR, newly synthesized DNA strand is labeled with radioactive nucleotide.
o
2. Terminal transferase - In this method, a terminal transferase enzyme will label the probe at the ends to the last nucleotide of the probe. Probe is incubated with the labeled nucleotide and terminal transferasae will add the labeled nucleotide at the end. A partial purification with gel filtration column will give labeled primer.

o
• Preparations of replica plate – As original genomic or CDNA library is precious and will be consumed in later stage, all procedure with the replica plate cont6aining clones in an identical manner.
o
• Blotting- The clone is transferred on a nitro cellulose membrane with retaining identical pattern of colonies on master plate. The cells on the membrane are lysed and released DNA is denatured, deprotinated and allowed to bind the membrane.
o
• Hybridization- A labeled probe prepared in step 1 will be added. Probe will bind to the target DNA due to base pairing. The membrane to remove unbound probe.
o
• Development of blot (Autoradiography)- The position of  labeled probe is detected by autoradiogram. The position of signal on membrane can be matched with the master plate to get location of the corresponding column.

Germplasm or genetic resources are stored in the form of seeds in a specialized place known as gene bank.

 

Gene banks are a type of bio repository which preserve genetic material.

For plants this is done by in-vitro storage, freezing cuttings from the plants or stocking the seeds.

PROCEDURE OF MAKING cDNA LIBRARY-

A cDNA library is a combination of clone cDNA (complimentary DNA) fragments inserted into a collection of host cells, of which constitute some portion of the transcripton of the organism and are stored as a library.

cDNA is produced from fully transcribed mRNA found in the nucleus and therefore, contains only the expressed genes of an organism.

A cDNA library represents mRNA population present at a particular stage in an organism into multiple clones containing small DNA fragments. It has following steps:

o
• Isolation of mRNA – The structure of typical mRNA is given below:

It has a CAP structure at 5’ , coding sequence and a polyA tail at its 3’ region.

The nucleotide A forms 2 hydrogen bonding with nucleotide T and this pairing is very specific. Exploiting this feature, mRNA population can be isolated from RNA pool, using poly-T affinity column.

The steps in mRNA isolation from cell is given below:

o
• Release of total RNA either by a lysis buffer containing detergent.
o
• Mixing of poly-T containing beads with the total RNA preparation. Due to mutual exclusive affinity, mRNA binds to the poly-T beads.
o
• Wash the beads with washing buffer to remove non specific, cross contaminating species.
o
• In new to the mRNA from beads, its purity can be checked from poly acrylamide gel.

 

o
• Preparation of complementary DNA- Multiple approaches have been developed to prepare complementary DNA (cDNA) from isolated  mRNA. In all approaches the three steps are perfomed.

o
• First strand synthesis with a reverse transcriptase.
o
• Removal of RNA template.
o
• Second strand synthesis.

 

         Homopolymer tailing:-

This method exploits the presence of poly-A tail present on mRNA to synthesize first DNA strand followed by degradation of RNA template and synthesis of second strand. It has following steps:

o
1. An oligo dT primer is used with mRNA as a template to prepare first strand of  DNA with the help of reverse transcriptase and dNTPs.
o
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.
o
3. DNA:RNA hybrid is loaded on an alkaline sucrose gradient. This step will hydrolyze RNA and allow the full recovery of cDNA.
o
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.

o
• Cloning of cDNA into the vector- The cDNA is ligated into the suitable vector to generate clone.
o
• 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.

 

 

ADVANTAGES OF cDNA LIBRARIES-

o
• Ease of studying the genome of different organisms.
o
• Identification of genes in an organism.
o
• Creation of transgenic organism.
o
• Gene therapy.
o
• Pharmaceutical Applications through the study of genetic defects.

 

 

 

CONSTRUCTION OF GENOMIC LIBRARY-

Gene sequences are arranged in genome in a random fashion and selecting or isolating is a big task especially when the genomic sequences are not known.

A small portion of genome is transcribed to give mRNA where a major portion  remains untranscribed.

PREPARATION OF GENOMIC LIBRARY-

A genomic library represents complete genome in multiple clones, containing small DNA fragments. Depending upon the organism and size genome, this library is either prepared in a bacterial vector or in a yeast artificial chromosome (YAC). It has following steps:

o
• Isolation of genomic DNA- Isolation of genomic DNA has following steps:

o
• Lysis of cells with detergent containing lysis buffer.
o
• Incubation of cells with digestion buffer containing protease-

K, SDS to release genomic DNA from DNA-protein complex.

o
• Isolation of genomic DNA by absolute alcohol precipitation.

o
• Purification of genomic DNA with phenol : chloroform  mixture. In this step, phenol denatures the remaining proteins and keep the protein in oragnic phase.
o
• Genomic DNA present in aq. phase  is again precipitated with absolute alcohol.
o
• Genomic DNA is analyzed on 0.8% agarose gel and a good preparation of genomic DNA give an intact band with no visible smear.

 

o
• Generation of suitable size fragments- Next step generation of genomic DNA into suitable size fragments.

             Restriction digestion-

    Genomic DNA can be digested with a fragment DNA cutting enzymes such as EcoR-I or BamH-I to generate the random sizes of DNA fragments. As fragments are randomly generated and are relatively big enough, it is likely that each and every genomic sequence is presented in a 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. The probability (P) of finding a particular genomic sequence in a random library of  N  independent clone is as follows:

         N = In (1-P)/ In (1-1/n)........ (Eq. 10.1)

o
• 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 of the vector (Table 10.1).

                    Size of average fragment can be calculated from the Eq. 10.1 and accordingly a suitable vector can be chosen.

o
• In the case of fragment generated by restriction enzyme, vector can be digested with the same enzyme and put for ligation to get clone.
o
• In the case of mechanical shearing mediated fragment generation putting these fragment needs additional effort.
o
• In one of the approaches, a adopter molecule can be used to generate sticky ends, alternatively a endonuclease can be used to generate sticky ends.

o
• 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.

 

METHODS OF SCREENING cDNA & GENOMIC LIBRARIES-

In a typical experiment, host bacteria containing either a plasmid-based or bacteriophage- based library are plated out on a petri-dish and allowed to grow overnight to form colonies.

         A replica of a bacterial colonies is then obtained by overlaying the plate with a nitrocellulose disc. The disc is removed, treated with alkali to dissociate bound DNA duplexes into single-stranded DNA, dried, and placed in a sealed bag with labelled probe. If the probe DNA is duplex DNA, it must be denatured by heating at 70C.

The probe & the target DNA complementary sequences must be in a single stranded form if they are to hybridize with one another. Any DNA sequences complementary to the probe DNA will be revealed by autoradiography of nitrocellulose disc.

                                Bacterial colonies containing clones bearing target DNA are identified on the film and can be recovered from master plate.

 

SCREENING BY DNA HYBRIDIZATION-

DNA sequence information can be exploited with a general rule that nucleotide present in a DNA sequence provides a specificity due to unique base pairing preferance of nucleotides.

                             A=T                         G=C

As a result a particular DNA sequence can be identified by a complementary single stranded sequence.

The DNA sequence used for this purpose is called ‘Probe’. The position of probe is the actual site of desirable clone of containing specific sequence. It has following steps:

o
• Preparation of radioactive probe- There are two different method used to label a single stranded DNA probe either at terminal or through out the sequence.

o
1. Random primer method- In this method, a random primer is used to anneal to the template and then a PCR reaction is performed in the presence of radio labeled nucleotide. After PCR, newly synthesized DNA strand is labeled with radioactive nucleotide.
o
2. Terminal transferase - In this method, a terminal transferase enzyme will label the probe at the ends to the last nucleotide of the probe. Probe is incubated with the labeled nucleotide and terminal transferasae will add the labeled nucleotide at the end. A partial purification with gel filtration column will give labeled primer.

o
• Preparations of replica plate – As original genomic or CDNA library is precious and will be consumed in later stage, all procedure with the replica plate cont6aining clones in an identical manner.
o
• Blotting- The clone is transferred on a nitro cellulose membrane with retaining identical pattern of colonies on master plate. The cells on the membrane are lysed and released DNA is denatured, deprotinated and allowed to bind the membrane.
o
• Hybridization- A labeled probe prepared in step 1 will be added. Probe will bind to the target DNA due to base pairing. The membrane to remove unbound probe.
o
• Development of blot (Autoradiography)- The position of  labeled probe is detected by autoradiogram. The position of signal on membrane can be matched with the master plate to get location of the corresponding column.