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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 ( [link] ).

In part A, the figure shows a strand of ladder-like DNA. In part B, the DNA is cut on both strands between the two guanines. In part C, the 2 strands have separated, leaving complementary sticky ends on each with unattached 5' to 3' G, A, T, and C nucleotides.
In this (a) six-nucleotide restriction enzyme recognition site, notice that the sequence of six nucleotides reads the same in the 5' to 3' direction on one strand as it does in the 5' to 3' direction on the complementary strand. This is known as a palindrome. (b) The restriction enzyme makes breaks in the DNA strands, and (c) the cut in the DNA results in “sticky ends”. Another piece of DNA cut on either end by the same restriction enzyme could attach to these sticky ends and be inserted into the gap made by this cut.

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 ( [link] ).

An illustration showing the steps in creating recombinant DNA plasmids, inserting them into bacteria, and then selecting only the bacteria that have successfully taken up the recombinant plasmid. The steps are as follows: both foreign DNA and a plasmid are cut with the same restriction enzyme. The restriction site occurs only once in the plasmid in the middle of a gene for an enzyme (lacZ). The restriction enzyme leaves complementary sticky ends on the foreign DNA fragment and the plasmid. This allows the foreign DNA to be inserted into the plasmid when the sticky ends anneal. Adding DNA ligase reattaches the DNA backbones. These are recombinant plasmids. The plasmids are combined with a culture of living bacteria. Many of the bacteria do not take any plasmids into their cells, many take plasmids that do not have the foreign DNA in them, and a few take up the recombinant plasmid. The bacteria that take up the recombinant plasmid cannot make the enzyme from the gene that the fragment was inserted into (lacZ). They also carry a gene for resistance to the antibiotic ampicillin, which was on the original plasmid. To find the bacteria with the recombinant plasmid, the bacteria are grown on a plate with the antibiotic ampicillin and a substance that changes color when exposed to the enzyme produced by the lacZ gene. The ampicillin will kill any bacteria that did not take up a plasmid. The color of the substance will not change when the gene for lacZ contains the foreign DNA insert. These are the bacteria with the recombinant plasmid that we want to grow.
This diagram shows the steps involved in molecular cloning.

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.

Questions & Answers

distinguish between properties and characteristics
Elee Reply
what are organelles
Elee
what is biology
Prevail Reply
biology is science that studies life
Elee
it's the study of living and non living things
now that we have an estimate for the diameter of the cell.what estimate can we make about the volume of the cell?
faxhood Reply
why too much insulin result in low blood sugar
Leri Reply
For example, too much exercise can cost you to lose to much weight. Too much insulin will pull to much sugar out of your systemic system into your cells.
Eric
difference between DNA and RNA
Eyitayo Reply
DNA is deoxyribonuclaic acid. Deoxy refers to a lack of oxygen. The Ribose moity is missing an OH group. I think it is missing from the second C of the ring. RNA is ribonucleic acid. DNA has our genetic code in on it. RNA is translated from DNA and carries the blue print for protein synthesis.
Eric
The OH group on RNA prevents it from being reactive. But it is very unstable though. Would you want such a power tool floating around in your body, no. And you have three types of RNA: mRNA, tRNA and rRNA. Please let me know it this helped?😄
Eric
o
Browse
Eric answered this question perfectly
what determines the aeration level in the soil
Shola Reply
what is homeostasis?
Sarita Reply
It means balance in a biological system.
Eric
What is biology
Don Reply
Biology z the study of life
GOLDEN
what's biology
buran
biology is the study of living nd none living organism
Chinaza
Biology is the study of life
Quadri
yes Sir
Said
what's cell biology
Prince
biology is the study of life
Raheal
what is asexual reproduction,?
Awoi Reply
A type of reproduction which does not involve the fusion of gametes or a change in the number of chromosomes
Serena
Reproduction without sex... In which form a single organism or cell makes a copy of itself.
Serena
Please explain the concept of mitosis and meiosis
Serena
I guess you could use it for study buddies and brushing up on what you need to
Serena
what is mitosis
Klp
Asexual reproduction?
Serena
why pepsin and trypsin released in active form?
Yacqub
mitosis is the type cell division in which two daughter cells have same no. of chormosomes
syed
Hi
Don
chromosome number remains the same in mitosis
mc
Ello
Henry
Hii
Nikky
Yrr help me.
Nikky
Physical chemistry..... Koi h jo mujhe physical chem ki notes send kr ske
Nikky
what is asexual reproduction
Targbe
what makes golgi body in plants
Abdulkareem Reply
name the membrane of the plants
Abdulkareem
how can turners syndrome be corrected before birth
Balinda
which animal survive from being preyed just because of being humble, slow, and not aggressive
Balinda
Plants have golgi body's also. Plants are eukaryotic cells. And membrane bound organelles are a characteristic of eukaryotic cells. Moreover golgi body's are creatted from the ER. Also do not forget plants have plastids and animal cells do not.
Eric
During organs transplantation, the organs cannot be taken from just anybody since the graft would be rejected sooner or later due to
Liter Reply
Non-MHC compatibility on the organ and an attack from the patient's immune system.
Eric
what makes golgi body in plants
Abdulkareem
why trypsin and pepsin released in active form
Yacqub
Let us remember MHC'S on our cells. This is how our cells determine self from n o n s e l f. Transplanted tissue has to have a certain amount markers. These have to match to the recipiant's markers. Even with this, immunosuppresant medacine is prescribed to the recipient.
Eric
Even with these measures the body may still reject the transport. This can occur even after the recipient excepting the transport for some time.
Eric
what is integument system
Joy Reply
This system is our skin. This includes the skin lining our alimentary system which includes the tissue from our mouth to our anus. Our skin is our largest organ system. It is mostly made up of epithelial tissue.
Eric
Cellular respiration
Lucy Reply
This is how our cells make energy. They use glucose + oxygen. There are other facors involves also. But these are the main two reactant used, for aerobic respiration. The main product is ATP. ATP is a high energy molecule which is paramount for life.
Eric
what are the characteristics of living things
Ruth Reply
Movement Respiration Nutrition/Feeding Irritability/Sensitivity Growth Excretion Reproduction Deat/Life span
Hashim
What makes children from the same father and mother sometimes don't look alike?
Hashim
One of the main one's is the abilit reproduce. That is why viruses are not considered living. Because they do not have the ability to replicate on they own.
Eric
identification of problems
Nana Reply
what happens in the process of raising the human arms
Nana

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Source:  OpenStax, Concepts of biology. OpenStax CNX. Feb 29, 2016 Download for free at http://cnx.org/content/col11487/1.9
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