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Which comes first, PCR or Gel electrophoresis?

Which comes first, PCR or Gel electrophoresis?


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If I want to find out wither a group of patients have the abnormal BCR-ABL cancer gene, how do I benefit from the PCR and the gel electrophoresis techniques? I'm kinda lost trying to determine which comes first and what is the exact function of each device, I have read the whole gene cloning process in my biology book but couldn't absorb it because it's so complex.


PCR comes first. If PCR works right, then a specific product will be amplified. But you cannot see it in the little plastic Eppendorf PCR tube. That is what the electrophoresis is for. After you run PCR, you run the products on an agarose gel to visualize it (with the help of some flourescent dye). The agarose gel is just to see if the PCR worked.


First PCR and Gel at Biomakespace

We rounded off the Michaelmas term with our first practical session at the newly opened Biomakespace! Our aim for the day was to test out the facilities at Biomakespace by running a PCR followed by performing DNA electrophoresis – both very important molecular biology techniques.

PCR, short for Polymerase Chain Reaction, is an extremely useful technique which enables us to massively amplify a specific region of a DNA template molecule. A commonly used template is genomic DNA extracted from tissues or cells, however we were using a selection of BioBricks left over from previous Cambridge iGEM teams. BioBricks are a selection of standardised ‘biological components’ which come inserted into larger circular DNA molecules called plasmids. BioBricks are typically distributed as 2-3 ng dry DNA samples in 384-well plastic plates which we dissolved in water on the day.

Since we plan to use Gibson assembly to build light-sensitive genetic circuits next term, we wanted to selectively isolate the sequence we need from the rest of the plasmid and to customise the ends. To do so, we needed to add carefully selected primers to the PCR samples. Last week (19/11/17) we had a meeting where we discussed some of the basics of good primer selection and designed a few set of primers predicted to amplify the sequences we wanted – thanks to Jarrod Shilts for helping us with this! Primers are small pieces of DNA, around 20 nucleotides long, which can be custom ordered from various companies. Ours came as tiny pellets of dried DNA in tubes which we dissolved and diluted to the working concentration. We had a few sets of primers: one set intended to bind and amplify directly from the start and finish of the coding regions of the BioBrick genes, and another two sets which amplified from common sequences shared between the plasmids our BioBricks were inserted into.

While our PCR was running (on a rather antique thermocycler) we set up the gel we were going to use for separating and visualising the DNA fragments we hoped to generate. DNA electrophoresis relies on the fact that DNA is negatively charged and thus will move from cathode (negative electrode) towards the anode (positive electrode) when placed in an electric field. The gel provides an environment which slows larger pieces of DNA more than smaller ones, which means that smaller ones migrate faster and thus DNA separates by size. By loading a marker sample, with several known-length pieces of DNA, one can estimate the size of DNA in other gel lanes (e.g. our PCR-amplified ones). We had to perform a little DIY to keep the liquid gel in the mould while it set which worked with only a little leakage. Just before pouring we added a dye which fluoresces when in contact with DNA. After running the gel, this dye enabled us to see our PCR products as bright bands when the fluorophore was excited by a blue light transilluminator.

The gel image isn’t as clear as one might like (partly due to a dodgy phone camera!) but there are very clear bands indicating that most of our PCRs were successful! Lane contents: the outer-most lane on each side contained a marker ladder, lanes 2-7 were samples amplified with primers directly over the gene coding regions (designed last week), lanes 8-13 were samples amplified with generic plasmid primers taken from the BioBrick repository website, lanes 14-19 were amplified with similar generic plasmid primers (also designed last week).

Great job to all who helped with the primer design and helped with the practical work! The results indicate that we have several sets of primers which will be useful as start-points for producing overhang primers required to prep sequences for Gibson assembly. There are a few lanes which produced unexpected results though. For example lanes 13 & 19, which used the same template, don’t show bands from either generic primer set. Conversely lanes 10 & 16 showed bands which look a bit larger than we anticipated. These are both things which may need investigating further – something one of our project managers may have time for over the vacation.

Thanks to everyone who has come to our events and meetings this term, we’re expecting things to get more exciting after the winter vacation as we attempt to assemble our light-sensitive circuits and boot them up in E. coli bacteria! Finally, many thanks to Katia Smith-Litière and especially Jenny Molloy for helping us with paperwork, ordering, and more which enabled us to get up and running at Biomakespace this term.


Gel electrophoresis

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Gel electrophoresis, any of several techniques used to separate molecules of DNA, RNA, or protein on the basis of their size or electric charge. Gel electrophoresis has a variety of applications for example, it is used in DNA fingerprinting and the detection of genetic variants and proteins involved in health and disease as well as in the detection and purification of nucleic acids and proteins for research. It is also used to aid in the detection of pathogens (disease-causing organisms) that may be present in blood or other tissues or in sources such as food. In many instances, nucleic acids or proteins that are detected and purified with gel electrophoresis are investigated further by means of DNA sequencing or mass spectrometry.

The gel electrophoresis apparatus consists of a gel, which is often made from agar or polyacrylamide, and an electrophoretic chamber (typically a hard plastic box or tank) with a cathode (negative terminal) at one end and an anode (positive terminal) at the opposite end. The gel, which contains a series of wells at the cathode end, is placed inside the chamber and covered with a buffer solution. The samples are then loaded into the wells with a pipette. The chamber is connected to a power supply that, when turned on, applies an electric field to the buffer. The electric field causes negatively charged molecules to migrate through the gel toward the anode. (DNA and RNA are negatively charged proteins must be treated with a detergent to give them a negative charge.) The molecules’ movement is influenced by the porous gel matrix such that larger, heavier molecules move relatively slowly, whereas smaller, lighter molecules move more quickly. The density of pores and the type of substance used to make the gel further influence the rate of molecule migration. Often a dyed “ladder,” or marker with multiple molecules of known and varying molecular weights, is run alongside experimental samples to serve as a reference for size. The dye enables the visualization of the marker as it moves through the gel samples typically are also dyed for visualization. A dye known as ethidium bromide, which fluoresces under ultraviolet light, frequently is used for crisp visualization of DNA samples.


Gel electrophoresis can be used to find genes associated with a disease

In this simulated case, the researchers are looking for DNA fragments that are only found in patients who have inflammatory bowel disease.

These DNA fragments are presented as ‘bands’ in the electrophoresis results (see the picture).

If the fragments are found only in people who have the disease, it suggests that the fragments contain the DNA from a gene variant that might mean a person is more susceptible to getting the disease.

The picture shows the results of DNA analysis from seven different people. The first lane (1) represents a ‘ladder’, which allows you to determine the size of the DNA fragments that have been separated.

Imagine that the DNA loaded in wells 2, 3, 4 and 5 comes from patients with the disease, and DNA loaded in wells 6, 7, 8 and 9 comes from people who do not have the disease. Can you see that in three of the four patients with the disease there is an extra band in the pattern?

The person with DNA loaded in lane 2 also has the disease, but the results do not show the same banding pattern as other people with the disease. This suggests that more than one genetic variation may be associated with this disease.


Help with PCR and Gel Electrophoresis. Not getting any bands - (Sep/07/2012 )


Can anyone help or advise me. I am a Student and have been trying to amplify cDNA produced from RNA unsuccessfully for a number of weeks now in order to complete a gene sequence where I have a small gap for around 40 nucleotides.
I performed the RT-PCR First Strand Synthesis using Fermatas Revert Aid Firrst Strand cDNA Synthesis Kit and after 10 attempts got a band using the following:
12ul MyTaq
1ul 18s Forward Primer
1ul 18s Reverse Primer
1ul concentrated cDNA
9.5ul Nuclease Free H2O

This was to check there was actually cDNA produced.
Primers were then designed from either side of the "missing segment" of around 45 nucleotides.

To amplify the gene in question (POR Gene) I have tried several things.
Initially I used:
16.25ul Nuclease Free H20
5ul Buffer
0.5ul dNTPs
1ul Fwd Primer
1ul Rev Primer
1ul Template
0.25ul Enzyme (Phusion Hotstart II)

PCR conditions used were
1 cycle:
98oC for 30 seconds
39 cycles of:
98oC 10 seconds
54oC 30 seconds
72oC 30 seconds
1 cycle:
72oC 5 minutes

I have now tried altering the following:
Changing to Velocity Enzyme
Seeded PCR
Halving the enzyme amount
Reducing extension time
Changing annealing temp from 54oC to 50oC and also 45oC
Adding more Template
Increasing annealing time to 60 seconds

All of the above has produced no bands at all, or smears, or the PCR product still in the wells after gel was ran.

I am really stumped now and would appreciate some advice.
I hope I have included everything I need in this post.

Many Thanks in advance.

How were your primers designed? Anything special about the template (high GC, low GC?, structure)?

I already have 2 sequences, one is the 5' end of the gene, and the other the 3' end. There is however a gap of around 45 nucleotides in the middle which is what I am trying to amplify. The primers were designed from these sequences either side of the gap, are 20 nucleotides long and not particularly GC rich.

Those look like perfectly reasonable primers. How do you know the length of this gap? Are you able to amplify the known regions of your gene from the same template sample?
Perhaps the gap is really an intron, and you have been amplifying genomic DNA rather than cDNA.

Did a Blast search and the compared the 2 sequences as from same family, using ClustalW2 and got an idea of the length of the gap.

And, can you amplify the portions you have sequence for from your cDNA template?
You could amplify genomic DNA with the two primers you have. If you get a short result, then you will cover the gap. If you get a long result, you will know there is an intron. What is the end goal? Are you trying to make the gene? If so, then you can probably substitute one of the homologous sequences you have apparently found for the 9 aa that are missing.

The sequences I already have were given to us along with several others from another research dept and when we did a BLAST search of them all in the database, the two we have are the ones that came up as the POR Gene but there is a small sequence missing in between them.

So, I would recommend that you try to amplify the sequences you already know are present. This will check that your cDNA (or perhaps genomic DNA) really is present and of sufficient quality. I would also try dilutions of your template.

I have already tried dilutions of the template, but will try the sequences I already know, thank you


Which comes first, PCR or Gel electrophoresis? - Biology

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Agarose extracted from sea weeds finds use in (AIPMT Pre. - 2011)
1. Spectrophotometry
2. Tissue Culture
3. PCR
4. Gel electrophoresis

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Which one of the following techniques made it possible to genetically engineer living organism? (AIPMT Mains-2011)
1. Recombinant DNA techniques
2. X-ray diffraction
3. Heavier isotope labelling
4. Hybridization

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For transformation, micro-particles coated with DNA to be bombarded with gene gun are made up of:(AIPMT Pre. 2012)
1. Silver or Platinum
2. Platinum or Zinc
3. Silicon or Platinum
4. Gold or Tungsten

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Which one is a true statement regarding DNA polymerase used in PCR (AIPMT Pre. 2012)
1. It is used to ligate introduced DNA in recipient cell
2. It serves as a selectable marker
3. It is isolated from a virus
4. It remains active at high temperature

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PCR and Restriction Fragment Length Polymorphism are the methods for: (AIPMT Pre. 2012)
1. Study of enzymes
2. Genetic transformation
3. DNA sequencing
4. Genetic Fingerprinting

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The figure below is the diagrammatic representation of the E.Coli vector pBR 322. Which one of the given options correctly identifies its certain component (s)? (AIPMT Pre. 2012)

1. ori - original restriction enzyme
2. rop-reduced osmotic pressure
3. Hind III, EcoRI - selectable markers
4. amp R , tet R - antibiotic resistance genes

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The figure below shows three steps (A, B, C) of Polymerase Chain Reaction (PCR). Select the option giving correct ‘identification together with what it represents?              (AIPMT Mains. - 2012)   

Options:
1. B - Denaturation at a temperature of about 98ଌ separating the two DNA strands.
2. A - Denaturation at a temperature of about 50ଌ
3. C - Extension in the presence of heat stable DNA polymerase
4. A - Annealing with two sets of primers


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NanoArmoring of Enzymes: Rational Design of Polymer-Wrapped Enzymes

Caterina M. Riccardi , . Challa V. Kumar , in Methods in Enzymology , 2017

3.1 Enzyme Conjugation to PAA and Characterization by Agarose Gel Electrophoresis

Agarose gel electrophoresis was done to verify complete conjugation of the enzymes to PAA in solution ( Fig. 1 A ). The nanoarmored GOx–HRP–PAA (Lane 3) showed increased electrophoretic mobility toward the positive electrode when compared to GOx/HRP (Lane 1) and GOx/HRP/PAA (Lane 2) controls. The increased negative charge is due to the covalent attachment of the enzyme (NH2 groups) to the negatively charged PAA (COOH groups), which also indicates that no unconjugated enzyme remained after the EDC reaction was complete ( Scheme 3 ). Normally EDC reaction is 40%–50% complete, but we achieved complete conjugation because of the possible optimal ratio of the enzymes to the polymer and high EDC concentration and conditions followed. Another reason is interlocking itself is expected to be efficient because not 100% of the available carboxyls or amine groups need to react. A substantial fraction should react to securely interlock the components in the fibrous matrix. Thus, there is a significant margin of error that is allowed in the extent of crosslinking needed for complete interlocking of the components.

Fig. 1 . (A) Agarose gel of the GOx–HRP–PAA conjugate in solution (Lane 3, concentration ratio of 16:3:10,000 μM GOx:HRP:PAA), corresponding controls of GOx/HRP (Lane 1, simple mixture in solution at a concentration ratio of 16:3 μM GOx:HRP) and GOx/HRP/PAA physical mixture in solution prior to EDC treatment (Lane 2). (B) SDS-PAGE of the GOx–HRP–PAA conjugate (Lane 4) shows the increased molecular weight compared to the corresponding controls of GOx/HRP (Lane 2) and GOx/HRP/PAA physical mixtures (Lane 3). Standard molecular weight markers are shown (Lanes 1 and 5).

Reproduced from Riccardi, C. M., Mistri, D., Hart, O., Anuganti, M., Lin, Y., Kasi, R. M., &amp Kumar, C. V. (2016). Covalent interlocking of glucose oxidase and peroxidase in the voids of paper: Enzyme–polymer “spider webs”. Chemical Communications, 52, 2593–2596.

Scheme 3 . Chemical reaction between amine and carboxyl groups catalyzed by EDC.

Prepare the agarose gel (0.125 g agarose) by microwaving a solution of agarose (0.5%, w/v) in Tris-acetate buffer (40 mM, pH 7.0, 25 mL) for 1 min on high setting. After microwaving, the agarose should all have dissolved.

Leave the agarose to cure in the gel mold for 30 min.

Meanwhile, prepare samples by mixing 5–6 μM with loading buffer (10 μL, 50% (v/v) glycerol, and 0.1% (w/v) bromophenol blue).

Load 15 μL of sample in each well. Once loaded, run the agarose gel at 100 V for 30 min.

Stain the gel with brilliant blue R250 (0.1%, w/v) for 4 h, and then destain the gel with acetic acid (10%, v/v) overnight.


10.1 Cloning and Genetic Engineering

Biotechnology is the use of artificial methods to modify the genetic material of living organisms or cells to produce novel compounds or to perform new functions. Biotechnology has been used for improving livestock and crops since the beginning of agriculture through selective breeding. Since the discovery of the structure of DNA in 1953, and particularly since the development of tools and methods to manipulate DNA in the 1970s, biotechnology has become synonymous with the manipulation of organisms’ DNA at the molecular level. The primary applications of this technology are in medicine (for the production of vaccines and antibiotics) and in agriculture (for the genetic modification of crops). Biotechnology also has many industrial applications, such as fermentation, the treatment of oil spills, and the production of biofuels, as well as many household applications such as the use of enzymes in laundry detergent.

Manipulating Genetic Material

To accomplish the applications described above, biotechnologists must be able to extract, manipulate, and analyze nucleic acids.

Review of Nucleic Acid Structure

To understand the basic techniques used to work with nucleic acids, remember that nucleic acids are macromolecules made of nucleotides (a sugar, a phosphate, and a nitrogenous base). The phosphate groups on these molecules each have a net negative charge. An entire set of DNA molecules in the nucleus of eukaryotic organisms is called the genome. DNA has two complementary strands linked by hydrogen bonds between the paired bases.

Unlike DNA in eukaryotic cells, RNA molecules leave the nucleus. Messenger RNA (mRNA) is analyzed most frequently because it represents the protein-coding genes that are being expressed in the cell.

Isolation of Nucleic Acids

To study or manipulate nucleic acids, the DNA must first be extracted from cells. Various techniques are used to extract different types of DNA (Figure 10.2). Most nucleic acid extraction techniques involve steps to break open the cell, and then the use of enzymatic reactions to destroy all undesired macromolecules. Cells are broken open using a detergent solution containing buffering compounds. To prevent degradation and contamination, macromolecules such as proteins and RNA are inactivated using enzymes. The DNA is then brought out of solution using alcohol. The resulting DNA, because it is made up of long polymers, forms a gelatinous mass.

RNA is studied to understand gene expression patterns in cells. RNA is naturally very unstable because enzymes that break down RNA are commonly present in nature. Some are even secreted by our own skin and are very difficult to inactivate. Similar to DNA extraction, RNA extraction involves the use of various buffers and enzymes to inactivate other macromolecules and preserve only the RNA.

Gel Electrophoresis

Because nucleic acids are negatively charged ions at neutral or alkaline pH in an aqueous environment, they can be moved by an electric field. Gel electrophoresis is a technique used to separate charged molecules on the basis of size and charge. The nucleic acids can be separated as whole chromosomes or as fragments. The nucleic acids are loaded into a slot at one end of a gel matrix, an electric current is applied, and negatively charged molecules are pulled toward the opposite end of the gel (the end with the positive electrode). Smaller molecules move through the pores in the gel faster than larger molecules this difference in the rate of migration separates the fragments on the basis of size. The nucleic acids in a gel matrix are invisible until they are stained with a compound that allows them to be seen, such as a dye. Distinct fragments of nucleic acids appear as bands at specific distances from the top of the gel (the negative electrode end) that are based on their size (Figure 10.3). A mixture of many fragments of varying sizes appear as a long smear, whereas uncut genomic DNA is usually too large to run through the gel and forms a single large band at the top of the gel.

Polymerase Chain Reaction

DNA analysis often requires focusing on one or more specific regions of the genome. It also frequently involves situations in which only one or a few copies of a DNA molecule are available for further analysis. These amounts are insufficient for most procedures, such as gel electrophoresis. Polymerase chain reaction (PCR) is a technique used to rapidly increase the number of copies of specific regions of DNA for further analyses (Figure 10.4). PCR uses a special form of DNA polymerase, the enzyme that replicates DNA, and other short nucleotide sequences called primers that base pair to a specific portion of the DNA being replicated. PCR is used for many purposes in laboratories. These include: 1) the identification of the owner of a DNA sample left at a crime scene 2) paternity analysis 3) the comparison of small amounts of ancient DNA with modern organisms and 4) determining the sequence of nucleotides in a specific region.

Cloning

In general, cloning means the creation of a perfect replica. Typically, the word is used to describe the creation of a genetically identical copy. In biology, the re-creation of a whole organism is referred to as “reproductive cloning.” Long before attempts were made to clone an entire organism, researchers learned how to copy short stretches of DNA—a process that is referred to as molecular cloning.

Molecular Cloning

Cloning allows for the creation of multiple copies of genes, expression of genes, and study of specific genes. To get the DNA fragment into a bacterial cell in a form that will be copied or expressed, the fragment is first inserted into a plasmid. A plasmid (also called a vector in this context) is a small circular DNA molecule that replicates independently of the chromosomal DNA in bacteria. In cloning, the plasmid molecules can be used to provide a "vehicle" in which to insert a desired DNA fragment. Modified plasmids are usually reintroduced into a bacterial host for replication. As the bacteria divide, they copy their own DNA (including the plasmids). The inserted DNA fragment is copied along with the rest of the bacterial DNA. In a bacterial cell, the fragment of DNA from the human genome (or another organism that is being studied) is referred to as foreign DNA to differentiate it from the DNA of the bacterium (the host DNA).

Plasmids occur naturally in bacterial populations (such as Escherichia coli) and have genes that can contribute favorable traits to the organism, such as antibiotic resistance (the ability to be unaffected by antibiotics). Plasmids have been highly engineered as vectors for molecular cloning and for the subsequent large-scale production of important molecules, such as insulin. A valuable characteristic of plasmid vectors is the ease with which a foreign DNA fragment can be introduced. These plasmid vectors contain many short DNA sequences that can be cut with different commonly available restriction enzymes . Restriction enzymes (also called restriction endonucleases) recognize specific DNA sequences and cut them in a predictable manner they are naturally produced by bacteria as a defense mechanism against foreign DNA. Many restriction enzymes make staggered cuts in the two strands of DNA, such that the cut ends have a 2- to 4-nucleotide single-stranded overhang. The sequence that is recognized by the restriction enzyme is a four- to eight-nucleotide sequence that is a palindrome. Like with a word palindrome, this means the sequence reads the same forward and backward. In most cases, the sequence reads the same forward on one strand and backward on the complementary strand. When a staggered cut is made in a sequence like this, the overhangs are complementary (Figure 10.5).

Because these overhangs are capable of coming back together by hydrogen bonding with complementary overhangs on a piece of DNA cut with the same restriction enzyme, these are called “sticky ends.” The process of forming hydrogen bonds between complementary sequences on single strands to form double-stranded DNA is called annealing . Addition of an enzyme called DNA ligase, which takes part in DNA replication in cells, permanently joins the DNA fragments when the sticky ends come together. In this way, any DNA fragment can be spliced between the two ends of a plasmid DNA that has been cut with the same restriction enzyme (Figure 10.6).

Plasmids with foreign DNA inserted into them are called recombinant DNA molecules because they contain new combinations of genetic material. Proteins that are produced from recombinant DNA molecules are called recombinant proteins . Not all recombinant plasmids are capable of expressing genes. Plasmids may also be engineered to express proteins only when stimulated by certain environmental factors, so that scientists can control the expression of the recombinant proteins.

Reproductive Cloning

Reproductive cloning is a method used to make a clone or an identical copy of an entire multicellular organism. Most multicellular organisms undergo reproduction by sexual means, which involves the contribution of DNA from two individuals (parents), making it impossible to generate an identical copy or a clone of either parent. Recent advances in biotechnology have made it possible to reproductively clone mammals in the laboratory.

Natural sexual reproduction involves the union, during fertilization, of a sperm and an egg. Each of these gametes is haploid, meaning they contain one set of chromosomes in their nuclei. The resulting cell, or zygote, is then diploid and contains two sets of chromosomes. This cell divides mitotically to produce a multicellular organism. However, the union of just any two cells cannot produce a viable zygote there are components in the cytoplasm of the egg cell that are essential for the early development of the embryo during its first few cell divisions. Without these provisions, there would be no subsequent development. Therefore, to produce a new individual, both a diploid genetic complement and an egg cytoplasm are required. The approach to producing an artificially cloned individual is to take the egg cell of one individual and to remove the haploid nucleus. Then a diploid nucleus from a body cell of a second individual, the donor, is put into the egg cell. The egg is then stimulated to divide so that development proceeds. This sounds simple, but in fact it takes many attempts before each of the steps is completed successfully.

The first cloned agricultural animal was Dolly, a sheep who was born in 1996. The success rate of reproductive cloning at the time was very low. Dolly lived for six years and died of a lung tumor (Figure 10.7). There was speculation that because the cell DNA that gave rise to Dolly came from an older individual, the age of the DNA may have affected her life expectancy. Since Dolly, several species of animals (such as horses, bulls, and goats) have been successfully cloned.

There have been attempts at producing cloned human embryos as sources of embryonic stem cells. In the procedure, the DNA from an adult human is introduced into a human egg cell, which is then stimulated to divide. The technology is similar to the technology that was used to produce Dolly, but the embryo is never implanted into a surrogate mother. The cells produced are called embryonic stem cells because they have the capacity to develop into many different kinds of cells, such as muscle or nerve cells. The stem cells could be used to research and ultimately provide therapeutic applications, such as replacing damaged tissues. The benefit of cloning in this instance is that the cells used to regenerate new tissues would be a perfect match to the donor of the original DNA. For example, a leukemia patient would not require a sibling with a tissue match for a bone-marrow transplant.

Visual Connection

Why was Dolly a Finn-Dorset and not a Scottish Blackface sheep?

Genetic Engineering

Using recombinant DNA technology to modify an organism’s DNA to achieve desirable traits is called genetic engineering . Addition of foreign DNA in the form of recombinant DNA vectors that are generated by molecular cloning is the most common method of genetic engineering. An organism that receives the recombinant DNA is called a genetically modified organism (GMO). If the foreign DNA that is introduced comes from a different species, the host organism is called transgenic . Bacteria, plants, and animals have been genetically modified since the early 1970s for academic, medical, agricultural, and industrial purposes. These applications will be examined in more detail in the next module.

Concepts in Action

Watch this short video explaining how scientists create a transgenic animal.

Although the classic methods of studying the function of genes began with a given phenotype and determined the genetic basis of that phenotype, modern techniques allow researchers to start at the DNA sequence level and ask: "What does this gene or DNA element do?" This technique, called reverse genetics , has resulted in reversing the classical genetic methodology. One example of this method is analogous to damaging a body part to determine its function. An insect that loses a wing cannot fly, which means that the wing’s function is flight. The classic genetic method compares insects that cannot fly with insects that can fly, and observes that the non-flying insects have lost wings. Similarly in a reverse genetics approach, mutating or deleting genes provides researchers with clues about gene function. Alternately, reverse genetics can be used to cause a gene to overexpress itself to determine what phenotypic effects may occur.

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    Osama bin Laden: The Science of His End

    "DNA is a charged molecule. Consequently, DNA molecules will move when an electrical field is applied to a liquid in which they are dissolved. If the liquid is a simple one--such as water with some salts in it--all the DNA molecules move at nearly the same speed. Under those conditions, it is hard to distinguish the tiny disparities in the motion of different kinds of DNA.

    "If the solution is made less liquid, as in a gel, and the DNA molecules all start moving across the solution from some initial small volume--that is, from essentially the same staring point--then the molecules can move at perceptibly different speeds. Usually smaller DNA molecules move faster than larger ones. After a while, the molecules are separated by size. If the molecules fall into only a few discreet sizes, then bands (little rectangles) of DNA will appear in the gel. Each of these bands contains DNA strands of a specific size."

    [Editors note: DNA fingerprinting uses gel electrophoresis to distinguish between samples of the genetic material. The human DNA molecules are treated with enzymes that chop them at certain characteristic points, thereby reducing the DNA to a collection of more manageably sized pieces. The DNA fragments are loaded into a gel and placed in an electrical field, which electrophoretically sorts the DNA fragments into various bands. These bands can be colored with a radioactive dye to make them visible to imaging techniques.]

    "For individual people, the bands of DNA created through this process will have a pattern that is specific to the individual. Part of this pattern comes from the size of the DNA part of it comes from the sequence of the DNA of a specific size.


    Watch the video: Gel Electrophoresis (February 2023).