Recombinant Dna Technology Gene Cloning Biology Essay

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The technology entailing all processes of varying the genetic material of a cell to make it competent of performing the preferred functions, such as producing novel substances. (http://www.biology-online.org/dictionary/Genetic_engineering)." Restriction endonucleases are a part of the natural defence mechanisims of bacteria against incoming DNA". Christopher. H. (1995). Gene cloning and manipulation. London: Cambridge."The endonucleases cleave the DNA at specific sites.They are called endonucleases because they cleave DNA within the molecule and are called restriction endonucleases because they restrict their activity to'foreign' DNA". Alan.E.H.Emery. (1985).An introduction to recombinant DNA. London:Portsmouth,Avon. Agarose gel electrophoresis is used to separate and analyze the DNA. It is mostly used for the separation of a particular band. But for the agarose gel electrophoresis few steps such as isolation restriction, ligation and transformation are done. That is a particular cell is first isolated, restricted, lysed and then transformed and then a DNA is separated and is run under a gel electro photometer. The gel electro photometer contains 7wells, which are prepared by placing a comb in the gel which is prepared by using a buffer. The comb makes 8 wells in which each of the 7 wells are loaded with different materials in order to isolate the DNA and to by getting different bands. There are few faint bands seen in the wells, and some bands are missing and some DNA bands are present and some DNA bands are departed from the actual mobility. These bands are then measured from the wells loaded and a graph is drawn in order to measure the log value of the rDNA. In the bacterial transformation there are no colonies seen on the plates, it might be due to some impurity, but if there was any impurity more colonies would have appeared, so it can be assumed that instead of ampicillin resistant, by mistake tet resistant might have been used on the plates or might be because of improper amount of the broth taken to be spread and so the colonies might have not appeared. If the experiment was done in a perfect manner there would be ampicillin resistant colonies seen growing on the plates and also white colonies.

INTRODUCTION:

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A method that produces indefinite amounts of scarce biological product by introducing DNA secluded from animals or plants into bacteria and then harvesting the product from a bacterial colony, as human insulin produced in bacteria by the human insulin gene. (http://dictionary.reference.com/browse/genetic+engineering)

Recombinant DNA is made up of a base consisting of sugar, phosphate and one nitrogen base; there are four nitrogen bases adenine, thymine, guanine and cytosine. They are paired as AT and GC. It is a double helical structure. The sugar used in DNA is deoxyribose.DNA makes only the proteins and not actually the organism. It is first transcribed into mRNA and then mRNA is translated into protein and protein then forms an organism. Recombinant DNA is also referred to as "chimera". The name recombinant is because one strand of DNA is combined with another strand of DNA.

Double Helix Structure

STRUCTURE OF DNA

(Mathew kuure-kinsey and Beth Mc Cooey for Biochemical Engineering Fall 2000)www.rpi.edu/dept/chem-eng/Biotech-Enivorn/Projectsoo/rdna.html.

DNA is taken from the cells by rupturing the cell membrane in the presence of anti coagulants. For this plasmids are used, plasmids are the circular, extra-chromosomal, double stranded DNA, which are having self replicating capacity( Brown, 2006).

The experiment used for the isolation of plasmid DNA is Alkaline lysis method. The isolated samples were analysed by the Agarose Gel Electrophoresis process.

Alkaline lysis method: Plasmids can be secluded by a array of methods many of which rely on the differential denaturation and reannealing of plasmid DNA compared to chromosomal DNA. One commonly used procedure developed by Birnboim and Doly involves alkaline lysis. This scheme basically relies on bacterial lysis by sodium hydroxide and sodium dodecyl sulfate (SDS), followed by neutralization with a high concentration of low pH potassium acetate. This gives discriminatory precipitation of the bacterial chromosomal DNA and other high molecular weight cellular components. The plasmid DNA remains in suspension and is precipitated with isoproponal. (http://biotechmethods.blogspot.com/2009/08plasmid-dna extraction-using-alkaline.html).

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DH5 (alpha) is a bacterial strain, its taxonomy ID:-668369.Its inherited blast name is enterobacteria.Its genetic code is translation table 11(bacterial archeal and plant plastid)(medicine.net, taxonomic virology).

Lysozyme is an enzyme that hydrolyzes the 1,4beta links between N-acetylmuramic acid and N-acetyl glucosamine, and thus vicious to cell walls of certain bacteria. (spaceflight.nasa.gov/history/shuttle-mir/references/glossaries/science/sc-gloss-g m.html).

pUC19- It is used as a cloning host, usually for non coding sequences of DNA or for genes that pUC19 contains 3 open reading frames, all of which are in the same point of reference.(www.madsci.org/posts/archives/2003-05/1052159932.Mb.rhtml)

pUC19 MCS

SacI SmaI XbaI SbfI

EcoRI KpnI BamHI SalI PstI SphI HindIII

agtgAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGcgtaatcatggtcat

. . . . . . .

4OO 41O 42O 43O 44O 45O 46O

...S N S S P V R P D E L T S R C A H L S P T I M T M

lacZtranslational start.(http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/maps/pUC19_map.pdf).

 

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puc19 by koshiks.

www.flickr.com/photos/biotechnology/2978048923/

http://bio.classes.ucsc.edu/bio20L/info/content/molbio2/puc19.gif

bio.classes.ucsc.edu/.../molbio1/index.htm

The above picture is the map of pUC19

Restriction: EcoR1 is a restriction endonuclease found in Escherichia coli. It cleaves DNA at the following specific sequence:

http://www.massey.ac.nz/%7Ewwbioch/DNAprot/ECOR1/seq1.gif

EcoR1 i.e Escherichia coli RY13 recognition sequences and cleavage sites from 5' to 3' are G * A A T T C,the enzyme EcoR1 normally cuts at the sequence -GAATTC- but in the presence of glycerol ". Alan.E.H.Emery. (1985).An introduction to recombinant DNA.London:Portsmouth,Avon.

DNA ligase: An enzyme that can refurbish breaks in a strand of deoxyribonucleic acid (DNA) by synthesizing a bond flanked by adjacent nucleotides. Under some conditions the enzyme can unite together loose ends of DNA strands, and in some cases it can renovate breaks in ribonucleic acid (RNA). It serves as a catalyst. An enzyme that seals notch in the DNA helix joins Okazaki fragments together during DNA replication and is vital in RDNA technology for DNA cloning. (http://medical-dictionary.thefreedictionary.com/DNA+ligase). In molecular biology, DNA ligation is to combine the two strands of DNA. Ligation is a natural process followed in both DNA repair and DNA replication, hence it has been adopted by genetic engineering laboratories as it is used in recombinant DNA technology and applications. Ligase, which is an enzyme that joins the phosphodiester bond between 3'-hydroxyl group and 5'-phosphate group. It needs ATP energy and Mg+2 as cofactor to perform the reaction (Brown, 2007). A high temperature prevents hydrogen bonding, while a low temperature decreases the chance of catalysis to occur. ligation is now using in gene cloning process to join the DNA fragments and in recombinant DNA technology to ligate the plasmid DNA with foreign DNA.

Bacterial Transformation: The exchange of genetic material between strains of bacteria by the transfer of a fragment of naked DNA from a donor cell to a recipient chromosome. (http://medical-dictionary.thefreedictionary.com/bacterial DNA into living cells is known as transformation (Lodge, 2007). Bacterial transformation is a technique widely used in molecular biology. In this technique, only competent bacterial cells that is, cells which have the ability to uptake foreign are transfected with plasmid DNA carrying foreign gene. By using this process, large quantities of plasmid DNA carrying desire gene can be produced. By using X-gal and IPTG isopropyl-ß-D-thiogalactopyranoside, we can screen the transformed colonies.

http://faculty.plattsburgh.edu/donald.slish/figures/Bluescript.GIF

(http://faculty.plattsburgh.edu/donald.slish/Transformation.html)

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Gel electrophoresis: It is a technique used to detach strands of different lengths of DNA. Restriction enzymes can be used to cut DNA into precise sizes and run through an agar gel using electricity. The unknown DNA can then be compared to a known DNA sample to calculate approximately the lengths of the unknown DNA segments.

Electrophoresis Box

https://sites.google.com/a/luther.edu/genetics/_/rsrc/1235968606119/students/elise-desroches/gel-electrophoresis/288px-Gel_electrophoresis_apparatus.JPG

https://eapbiofield.wikispaces.com/file/view/288px-Gel_electrophoresis_apparatus.JPG

The aim of the experiment is to create a recombinant DNA.

MATERIALS AND METHODS:

50 mM glucose, 25 mM Tris-HCL, pH 8.0, 10 mM EDTA containing 4 mg cm-3 Lysozyme.

0.2 M NaOH, 1% SDS

3 M potassium acetate, pH 4.8

DH5a - It is a control strain.

DH5a [pUC19] - It contains the plasmid pUC19.

Follow the practical booklet from pages(6-16)

RESULTS:

Agarose gel electrophoresis:

Gel Electrophoresis Picture.jpg

In the above picture there are 7 wells, well 1 is loaded with DH5a extract, well 2 was loaded with DH5a[pUC19]extract, well 3 was loaded with #1 pUC19 +EcoR1,well 4 was loaded with #2pUC19 -EcoR1,well 5 was loaded with #3 λ +EcoR1,well 6 was loaded with λ +pUC19-ligase,well 7 was loaded with λ +pUC19+ligase.

Distance travelled from the well to the band

Log kb

1.7

1.338

2.1

0.876

2.4

0.758

2.6

0.681

3

0.533

The above are the distances calculated from the 3rd well to the bands

The above graph showing the log kb values on the x-axis and the distance travelled on the y-axis.

TRANSFORMATION

Table. Colonies in the plates

Agar plates

Components

Blue colonies

White colonies

1

DH5α(-ve control)

0

0

2

PUC19

0

0

3

PUC19+λ-Ligase

0

0

4

PUC19+λ+ligase

0

0

5

One in 10 dilution

0

0

6

One in 10 dilution

0

0

Bacterial transformation: There were no colonies seen on the LB plates.

The below are the results of KISHORE MALABANTI,id no 08194614:

Table 1: Distance travelled by λDNA fragments and log size:

Distance travelled by λDNA fragments:

Log Kb values:

31

1.338

44

0.876

50

0.773

54

0.744

60

0.681

Figure: The graph shows that distance travelled by λDNA fragments.

The graph was plotted by taking distance travelled by the λDNA fragments on X-axis and

Log kb value on Log kb value on Y-axis. The distance travelled by the λDNA fragments was found at 65 with a kb value of 0.59.

The size of the plasmid DNA is 3.89 kb.

Bacterial transformation results:

Table3. Colonies in the plates

Agar plates

Components

Blue colonies

White colonies

1

DH5α(-ve control)

0

0

2

PUC19

3

0

3

PUC19+λ-Ligase

0

0

4

PUC19+λ+ligase

4

1

5

One in 10 dilution

0

0

6

One in 10 dilution

0

0

Results found were as follows:

Plate-1: No colonies were obtained.

Plate-2: 3 blue colonies were observed.

Plate-3: No results were obtained.

Plate-4: 4 blue colonies and 1 white colony were observed.

Total- 8 colonies were observed.

Calculations:

No, of white colonies = 1.

No. of blue colonies = 7.

Total no. of colonies = 8.

Formula:

Percentage of Transformation:

= No. of white colonies/Total no. of colonies Ã- 100

= 1/7Ã-100

= 14.2%.

Efficiency of Transformation:

Formula:

= No of transformed colonies Ã- 100

0.001

= 7 Ã-100

0.001

= 700000 µg -1 DNA.

DISCUSSION:

The 1st well loaded with DH5a extract had plasmid DNA without pUC19,to this stopping mixture was added as the extract wasn't sufficient,the stopping mixture was even added to the 2nd well sample i.e DH5a[pUC19]extract.for the 6th and the 7th well the stopping mixture was added to the sample tubes given for ligation,the whole sample was been used and so to load the wells the stopping mixture was added.and hence this might be the reason for the faint anomalies of DNA seen on the 6th and the 7th lanes loaded with + and - ligase.The bands seen in the 1st lane are faint and smeared. This might be due to insufficient quantity of DNA loaded on the gel or might be due to less time of electrophoresis or use of low voltage or higher percent gel or might be the DNA was degraded, might be the DNA was contaminated with protein. The bands seen in the 2nd lane are very faint which are invisible to the naked eye which might be due to improper W light source was used for visualization of ethidium bromide-stained DNA.

According to the protocol the 3rd well was loaded with restricted λ DNA.

There are 5 clear bands visible, whereas there should be 6 bands. And so we assume that the 3rd and the 4th bands are merged. And so we need to take the average of the 3rd and 4th band values average to plot them.

This well has been loaded with pUC19 which has many bands

And the pUC19 has settled down to the bottom and has a faint band visible clearly.

Due to some problem the bands have been merged together.

But we can make them out. The band is super coiled.

There are no bands visible in the 5th lane because of some impurity.it might be due to insufficient sample, or excessive or less amount of time given for incubation or freezing,or less amount of centrifugation time.or might be the sample was not loaded correctly in the well i.e till the whole well was filled.

There are no results for ligation.There are no results for ligation. It might be due to the insufficient amount of the sample

The well wasn't loaded. These situations can be avoided and taken care by avoiding nuclease contamination, by increasing/decreasing the amount of DNA as required or by using high amounts of gels,use of phenol extractions, use of ethanol precipitation (http://www.bio.davidson.edu/Courses/molbio/tips/trblDNAgel.html)

Bacterial transformation:

There were no colonies found on the plates. It might be due to tet resistant bacteria in ampicillin media. There should be actually colonies growing on the ampicillin media whereas the colonies might have not shown its presence because of a different resistant like tetracycline resistant,which donot grow colonies which actually have to grow on an ampicillin resistant media plate. The colonies might not have appeared on the plates because alkaline phosphatase was not used. The colonies on these plates will be blue in colour and there might also be break through colonies also called as satellite colonies,these colonies appear around the blue colonies,if there are any well separated white colonies it might be a recombinant most probably,or might be due to some kind of contamination,the reason for the colonies not appearing might also be due to overheating of the inoculating tube, or improper concentration of the sample.

CONCLUSION:

The aim of the experiment was not achieved.