Clonning

CLONING

1. Cell-based DNA cloning
2. Polymerase mediated in vitro DNA cloning

The importance of DNA cloning:

Current DNA technology is based on two different approaches:

a.   Specific amplification (DNA cloning) which involves cell-based DNA cloning (involving a vector/replicon and a suitable host cell) and in vitro DNA cloning (PCR)

b.   Molecular hybridization where the DNA fragment of interest is specifically detected using a mixture of different sequences

                      

Figure 1: DNA Clonning in vivo and In Vitro

Four steps in cell-based cloning:

1. Construction of recombinant DNA molecules. Involves the use of endonuclease restriction enzymes, ligation, and a replicon (vector).
2. Transformation in appropriate host cells.
3. Selective propagation of cell clones. This step takes advantage of selectable markers.
4. Isolation of recombinant DNA from cell clones followed by molecular characterization (such as restriction enzyme analysis).

Recombinant DNA Libraries (3 types):

1. Genomic library, Collection of cloned restriction enzyme digested DNAs containing at least one copy of every DNA sequence in a genome.
2. Chromosome library, Collection of cloned restriction enzyme digested fragments from individual chromosomes.
3. Complementary DNA (cDNA) library, Collection of clones of DNA copies made from mRNA isolated from cells.

·         reverse transcriptase (RNA dependent DNA polymerase)

·         Synthesizes DNA from an RNA template

·         cDNA libraries reflect what is being expressed in cells.

1. Genomic Library:

3 ways to make a genomic library:          

1. Complete digestion (at all relevant restriction sites)

1. Produces a large number of short DNA clones.
2. Genes containing two or more restriction sites may be cloned in two or more pieces.

2. Mechanical shearing

1. Produces longer DNA fragments.
2. Ends are not uniform, requires enzymatic modification before fragments can be inserted into a cloning vector.

3.      Partial digestion

1. Cut at a less frequent restriction site and limit the amount and time the enzyme is active.
2. Results in population of large overlapping fragments.
3. Fragments can be size selected by agarose electrophoresis.
4. Fragments have sticky ends and can be cloned directly.

Screening a genomic library (plasmid or cosmid):

1.      Plasmid vectors containing digested genomic DNA are transformed into E. coli and plated on selective medium (e.g., ampicillin).

2.      Colonies that grow are then are replicated onto a membrane (E. coli continues to grow on the membrane).

3.      Bacteria are lysed and DNA is denatured.

4.      Membrane bound DNA is next probed with complementary DNA (e.g., 32P radio-labeled DNA).

5.      Complementary DNA in the probe is composed of DNA sequence you are looking for; homologous sequence presumably also found in library.

6.      Unbound probe DNA is washed off the filter. 

7.      Hybridization of probed DNA is detected by exposure to X-ray film (or by chemiluminescence).

8.      Pattern is noted from exposure pattern of clones on X-ray film.

9.      Select clones that test positive and isolate for further analysis.

2. Chromosome Library:

1. Screening can be reduced if target genes can be localized to a particular chromosome.
2. Chromosomes can be separated by flow cytometry.

                                I.      Condensed chromosomes are stained with fluorescent dye.

                             II.      Chromosomes separate based on the level of binding of the dye and are detected with a laser.

3. cDNA Library:

1. cDNA is derived from mature mRNA, does not include introns.
2. cDNA may contain less information than the coding region.
3. cDNA library reflects gene activity of a cell at the time mRNAs are isolated (varies from tissue to tissue and with time).
4. mRNA degrades quickly after cell death, and typically requires immediate isolation (cryoprotectants can increase yield if immediate freezing is complicated by field work).

Creating a cDNA library:

Step 1-Isolate mRNA:

•         Mature eukaryote mRNA has a poly-A tail at the 3’ end.

•         mRNA is isolated by passing cell lysate over a poly-T column composed of oligo dTs (deoxythymidylic acid).

•         Poly-A tails stick to the oligo dTs and mRNAs are retained, all other molecules pass through the column.

Step 2-cDNA synthesis:

Step 3-Clone cDNA

•         Anneal a short oligo dT (TTTTTT) primer to the poly-A tail.

•         Primer is extended by reverse transcriptase 5’ to 3’ creating a mRNA-DNA hybrid.

•         mRNA is next degraded by Rnase H, but leaving small RNA fragments intact to be used as primers.

•         DNA polymerase I synthesizes new DNA 5’ to 3’ and removes the RNA primers.

•         DNA ligase connects the DNA fragments.

•         Result is a double-stranded cDNA copy of the mRNA.

Screening a cDNA library:

1. cDNA libraries are most often used to detect genes for proteins (cDNAs are generated for genes that are transcribed!).
2. If you know the DNA sequence for the protein coding gene you want to find, a homologous DNA probe can be used.
3. If no homologous DNA sequence is available, cDNA can be probed with an antibody that recognizes the protein.
4. Expression vector: cloned cDNA is inserted between a promoter and transcription terminator before it is transformed.
5. mRNA is transcribed from the cDNA and translated.
6. Colonies (now expressing proteins) are transferred to membrane.
7. Membrane is incubated with radioactive labeled antibody probe that recognizes the protein (non-radioactive chemiluminescent probes also are available).
8. Colonies with bound antibodies leave a dark spot on X-ray film.

                                      

  Figure 2: Methods of cDNA Clonning

Polymerase mediated in vitro DNA cloning:

Polymerase: DNA polymerase duplicates DNA. Before a cell divides, its DNA must be duplicated

Chain Reaction: The product of a reaction is used to amplify the same reaction. Thus, PCR results in rapid increase in the product.

PCR-Polymerase Chain Reaction is used to amplify a short, well-defined part of a DNA strand which is usually up to 10 kb. PCR, as currently practiced, requires several basic components such as:

—  DNA Polymerase, for amplifying DNA in vitro

—  DNA template, which contains the region of the DNA fragment to be amplified

—  Two primers, which determine the beginning and end of the region to be amplified DNA-Polymerase, which copies the region to be amplified

—  Nucleotides, from which the DNA-Polymerase builds the new DNA (dNTPs)

—  Buffer, which provides a suitable chemical environment for the DNA-Polymerase

—  Thin wall tubes and Thermal Cycler.

History:

—  Concept forward by H. G. Khorana et al. in 1971 ( before gene sequencing or a viable thermostable DNA polymerase)

—  15 years later Kary Mullis, coined PCR, and put into practice (Nobel Prize in Chemistry, October 1993)

—  In vitro DNA replication by DNA polymerase.

—  Mullis's original PCR process was very inefficient since it required a great deal of time, vast amounts of DNA-Polymerase, and continual attention throughout the PCR process.

—  Thermus aquaticus (Taq polymerase)

—  A disadvantage of Taq - sometimes makes mistakes (mutations) in the DNA sequence (no proofreading exonuclease activity)

—  Polymerases such as Pwo or Pfu, obtained from Archaea, have proofreading mechanisms

—  Combinations of both Taq and Pfu are available nowadays that provide both high fidelity and accurate amplification of DNA.

In vivo DNA replication requires many components such as DNA polymerase, DNA ligase, Primase, Helicase, Topoisomerase, Single strand binding protein etc for unwinding and synthesis of new copies of DNA. But in artificial DNA amplification, it is not possible to include all these enzymes because of their stability and regulation.

The PCR process consists of a series of twenty to thirty cycles. Each cycle consists of three steps;

      1. Denaturation

      2. Annealing

      3. Extension

Figure 3: Temperature graph for PCR 

1. Denaturation:

DNA denaturation is defined as the separation of double stranded DNA into two single stranded ones. In the PCR, Template DNA which is ds DNA (linear or circular) or cDNA (complementary DNA produced from produced mRNA by reverse transcriptase) is denatured by heating at 94-96°C. The heating in machine breaks apart the hydrogen bonds and separates apart. It should be remembered that an extended time of heating is required to ensure that both the template DNA and the primers have completely separated and are now single-strand only. Usually, 1-2 minutes heating is required for complete denaturation

 Figure 4: DNA Denaturation by heating at  above 90oC

2. Annealing

It is a primer binding step. Primers can attach themselves to the single DNA strands. Usually 5°C below their melting temperature (45-60°C) is required for annealing of the primers. A wrong temperature during the annealing step can result in primers not binding to the template DNA at all, or binding at random. The annealing time is usually 1-2 minutes.

Primers are the pairs of oligonucleotides each 18-25 nucleotides long. It should have 40%-60% GC content. The melting temperature of both should not differ by 5^oC. The 3’ terminal sequences of any primer should not be to any sequences of the other primer in the pair. The self-complimentary sequences (inverted repeats) of 3 bp is avoided in the primer. Two primers; forward primer and backward primer are used in the PCR.

What is the annealing temperature for the following primer (a 21 mer)?

                              3’-AAGCTTGTCCAGAATTTCGGC-5’

Solution: Every A and T nucleotide is responsible for 2 °C while G and C for 4 °C

      Therefore         (A+T) = 11 x 2 = 22

                              (C+G) = 10 x 4 = 40

      Now;

                                          22 + 40 = 62 °C (Melting temperature)

The annealing temperature is chosen as a few degrees below that number, so it is at about 57°C.

3. Elongation

It starts at the annealed primer and works its way along the DNA strand. The elongation temperature depends on the DNA-Polymerase. The time for this step depends both on the DNA-Polymerase itself and on the length of the DNA fragment to be amplified.

—  As a rule-of-thumb, 1 minute per 1000bp.

—  Elongation. Heat at 72°C for 45 seconds.

The DNA polymerase duplicates DNA in vivo which is necessary for reproduction of new cells. DNA strands are anti-parallel; One strand goes in 5' to 3' while the complementary strand is opposite (3' to 5' and dNTP follow standard base pairing rule). The DNA polymerase always moves in one direction (from 5’ à 3’) and incorporates the four nucleotides (A, T, G, C) to the growing chain. More than one DNA polymerases exist in different organisms. DNA polymerase needs Mg⁺⁺ as cofactor. Each DNA polymerase works best under optimal temperature, pH and salt concentration. During PCR, PCR buffer provides optimal pH and salt condition.

Taq DNA polymerase:

It is derived from Thermus aquaticus, a bacterium from hot stream. It is highly heat stable DNA polymerase which has an ideal temperature of 72°C. However the regular Taq DNA polymerase lacks 3’ - 5’ exonuclease activity needed to provide proof-reading function. Nowadays the Taq polymerase is available with proof reading function. The required temperature during DNA denaturation, annealing and extension is provided by PCR machine.

4. Repetition

Steps 2-4 are repeated 25-35 times, but with good primers and fresh polymerase, 15 to 20 cycles is sufficient. After complete cycles, the DNA of interest will be amplified to sufficient amount.  The machine itself is also used for storage of PCR products for certain period.

Applications:

—  Molecular biological technique for amplifying DNA without using a living organism, such as E. coli or yeast.

—  PCR is commonly used in medical and biological research labs for a variety of tasks, such as: 

—  Detection of hereditary diseases,

—  Identification of genetic fingerprints,

—  Diagnosis of infectious diseases,

—  Cloning of genes,

—  Paternity testing

Limitations:

The PCR technique has two limitations:

      1. Short sizes of amplified products (<5 b="" kb="">

      This is solved by doing Long-range PCR (up to tens of Kb long) which uses a mixture of two heat stable polymerases that provide optimal levels of DNA synthesis as well as a 3’ -> 5’ exonuclease activity.

     

      2. Low yields of amplifications

      Which is resolved by cloning the PCR amplified DNA fragment in a vector then propagating the vector in a cell based system (clone by A/T cloning or by using anchored PCR primers).

Advantages: Three major advantages of PCR are;

            1. Rapid

            (possible to amplify DNA from very quickly 2-3 hours than in cell based technique)

            2. Sensitive

            (possible to amplify DNA from a very small amount=pico mole of DNA)

            3. Robust

            (possible to amplify DNA from damaged tissues or degraded DNA)

Figure 5: Steps in DNA amplification by PCR