Restriction Enzyme digestion of DNA Exercise 8 Objectives

Restriction Enzyme digestion of DNA - Exercise 8 Objectives -Understand how Restriction Enzymes digest DNA. -Know how to construct a p. AMP (plasmid) or gel. -Given the size of fragments, gel, know how to construct a restriction map. -Given a restriction map know how to construct a gel.

NOTE: DNA IS Negatively charge because of the phosphate groups.

• DNA molecules are macromolecules that hold the genetic information of living organisms. They are extremely long, double-stranded polymers of nucleotides. • The covalent bond joining adjacent nucleotides in DNA is called a phoshodiester bond. • The phoshodiester bonds between nucleotides in DNA molecules are very stable unless they are physically stretched or exposed to enzymes name nucleases.

• Enzymes are capable of breaking (hydrolyzing) phoshodiester bonds in DNA molecules. Nucleases can be classified into two major groups: exonucleases and endonuclases. • Exonucleases: If the enzyme digest nucleotides from the ends of the DNA molecules. • Endonuclases: If the enzyme digest nucleotides in the interior of a DNA molecule. • Restriction endonuclease: An enzythem that digest DNA by recognizing specific short sequences of bases that are called palindromes.

• A special class of endonucleases from a bacteria has been isolated for this experiment. These special enzymes, termed restriction endonucleases (RE), digest DNA by breaking bonds only within a specific short sequence of bases. These base sequences usually ran in size from 48 base pairs but can be as long as 23 base pairs. • Restriction endonucleases confer an adaptive advantage on bacteria by digesting foreign DNA usually from an invading bacteriphage (bacterial virus). The resulting DNA fragments can then be further degraded and destroyed by exonucleases. These enzymes are used to cut DNA in a precise and predictable manner. They are extensively useful in gene cloning, DNA amplification, and many recombinant DNA technologies. • Restriction endonuclease (RE). This RE are also attained from bacteria. In a bacteria where we get these enzymes form there protected because if a virus invades a bacteria cell these endonuclease will chop up the virus DNA, its like a defense system, so we can isolate these endonuclease for experiments, but bacteria produce these endonuclease to protect themselves from foreign DNA entering their cells.

2 Restriction Endonucleases (RE) • Eco. R 1 & Hind. III. Both of these recognize different nucleotide sequences. • Each strand of DNA is cut at the phoshodiester bond between the G and A bases (indicated by the arrow signs). Notice that the sequence GAATTC is the same on both strands when each strand is read 5’ -> 3’. Such symmetrical sequences are called palindromes (In a English language a palindrome reads the same thing in both directions). This enzyme cuts the double strands asymmetrically, leaving protruding ends. These protruding bases are referred to as sticky ends aka compatible cohesive ends.

• Eco. R 1: Eco. R 1 recognizes palindrome on DNA, and cuts the bond between G & A, and G & A. When you do that it opens your DNA. For example, if you have plasmid and that palindrome is present once on the plasmid, you’ll get one cut. • If somewhere else that palindrome is present and you incubate it with Eco. R 1, you’ll get another cut. So every time Eco. R 1 recognizes this palindrome on your plasmid, it will cut through the DNA. So when it opens up the DNA, may get a couple of unpaired bases, and those unpaired bases are called sticky ends, and if you throw some nucleotides from different species, you can make recombinant DNA.

• Like Eco. RI, Hind. III also recognizes a palindromic sequence, AAGCTT, and produces sticky ends. Sticky ends can hydrogen bond together other because of complementary base pairing.

• Recombinant DNA molecules are compose of DNA fragments from two or more sources. Not all RE’s produce sticky ends. Some enzymes cut DNA to produce blunt ends, as shown here.

• Once the DNA has been digested, the fragments must be separated and identified. Fragments are separated by agarose gel electrophoresis. Agar is a large polysaccharide. • Gel electrophoresis: You put an agorose gel (agrose is a polysaccharide) and it has spaces, your DNA can move through these spaces, you put a current against this, the negative end is up, the positive is at the bottom, and because your DNA has a negative charge, the DNA moves down towards the positive end. • Gel is immersed in an ionic buffer. The buffer has a p. H above 8. 0 DNA at this p. H is negatively charge because the phosphates in the DNA backbone have lost hydrogen ions. The dye molecules serve as the indicator of the movements of invisible DNA molecule through the gel as an electric current is run through the gel. The negatively charged DNA will migrate from the anode to the cathode (negative to positive) along with the current.

• Separation of the DNA fragments occurs as they migrate through the network of agarose molecules. Smaller fragments slip through the network fast than large molecules. The rate of migration is a function of fragment size, as well as the density of agarose. The tightness (concentration of agarose). • High concentration favor smaller fragments. • Low concentration favor large fragments. • Each of migration function of fragment size and density of agarose. Depending on what conformation a circular DNA gets, it will run differently in the gel. So not only does the size of the DNA molecule affect migration rate, but the configuration of the DNA also affects the migration rate. The DNA that you will electrophoresing can exist in three different conformations.

• 1. Supercoil circular: 1 st fastest. When its circular it becomes twisted and turn and be comes a little bit shorter in size. Migrates fastest down the gel. Contains small volume, more compacted. • 2. Linear: Migrates next fastest down the gel. • 3. Nicked (relaxed) circular: One strand is intact, the other is broken and when it is nicked, it becomes extended. This one is very relaxed and faces the most difficulty making its way through the agarose. Supercoil < Linear < Nicked (relaxed) circular

• In addition to conformation affecting migration rate, laboratory production of plasmid DNA can be produce very large molecules that migrate very slowly. Two possible molecules that can be produced are dimers and concatemers. A dimer consists of two plasmids covalently linked in a series end to end. A Concatemer, for example, might consist of two plasmids with one hooked through the other but not covalently linked to each other. If a purified uncut plasmid is applied to a gel, bands of super coiled plasmid, nicked circular plasmid, dimers, and concatemer can be observed. • Dimer: Means that its link together by 2 links • Concatemer: Mean a whole bunch of plasmids linked together but not covalently linked to each other.

• p. AMP - the plasmid DNA. What we did in the experiment on DNA restriction analysis is we took p. AMP (circle) and incubated the p. AMP this plasmid with different restriction endonucleases. • From your electrophoresis gel, you can estimate the size of p. AMP. You can also determine if p. AMP is circular or linear. Finally, you can use the gel to draw a restriction map. A restriction map is a physical map of a piece of DNA showing recognition sites of specific restriction enzymes separated by lengths marked in numbers of bases. Separated DNA base on size • The pattern of DNA bands is characteristic for a specific DNA sample and the restriction enzymes used to cleave it. A banding pattern can be referred to as a DNA fingerprint. because it is unique to that particular DNA (and the combination of restriction fragments). • We ran a gel to see if we could determine how many DNA fragments you got. By electrophoresing a series of fragments of known size (DNA ladder) along with the DNA samples of interest, the sizes of unknown fragments can be estimated.

• A restriction site is a place where an enzymes cuts DNA, so there are restriction sites for Eco. R 1, and for Hind. III. • When constructing the p. AMP no restriction site where you start and where you finish. • Lane 4: A control to see what uncut plasmid looks like. How uncut DNA traveled whether they made 1 or 2 pieces. It’s your plasmid DNA was on tube 4 which acts like a measurements and acts like a ladder. No enzyme (Lane 4). • Lane 5: DNA ladder: DNA digest, containing known base pair lengths compare with fragments in lanes 1 -3. You will run DNAs of known size (DNA ladder) to help you estimate the size of your DNA fragments. Lane 5 contains DNAs of known sizes (DNA ladder).

Prokaryotic (Circular) DNA • DNA from bacteria (both chromosomal DNA and extra chromosomal plasmid DNA) and viruses is often a closed circle. If you have a circular DNA, we know that’s Prokaryotic DNA. In Prokaryotic DNA, the number of fragments will equal the number of restriction sites. Eukaryotic (linear) DNA • If you have one restriction site for an enzyme, you would have 2 fragments, and if you have 2 restriction sites for an enzyme, you would have 3 fragments. In Eukaryotic DNA, the number of fragments is always going to have one more or one less than restriction sites. • In Eukaryotic DNA, there’s no reason to see multiply bands in control lane because Eukaryotic DNA is linear, it doesn’t exist as supercoil, relax, or multimere so this is a hint in lane 4. So when you have Eukaryotic DNA, you will not see multiply bands in the control lane. • Also, just because they show you multiply bands, not every time your going to have prokaryotic (circular) DNA you get multiply lanes, its only if the DNA has been damaged into a supercoil.

What’s going to effect the movement of the DNA (Factors)?

• Size: small pieces migrate faster, farther than bigger pieces. • Conformation (shape): Comparing 3 pieces of DNA that are the same size. Supercoil < Linear < Nicked (relaxed) circular • Charge: Charge (+, -)DNA is negative because of Phosphate groups (anode) to positive (cathode).

Digestion of p. AMP with Eco. RI & Hind. III • We incubated our plasmid under several conditions. Those conditions were that we incubate p. AMP. • Lane 1: Eco. R 1 - one band • Lane 2: Hind. III - one band • Lane 3: Eco. R 1 & Hind. III - two bands • Lane 4: Water - Our control. We got one main band. • Lane 5: DNA ladder, a tool to measure the size of DNA fragments.

When constructing the p. AMP. There’s no restriction site where you start and where you finish the map. You could call this point the reference point. Also, all your base pairs (fragments) have to equal the total number base pairs of your plasmid. For example, 6, 000 Bp’s in this example.

Starting & Ending Point

Key for p. AMP KEY Enzyme A: Light green Enzyme B: Pink Enzyme C: Orange

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Are the number of fragments correct?

How do you cut and paste DNA? • Enzymes that cut DNA at specific short sequence sites – Restriction enzymes digest DNA • Blunt end cut • Asymmetric end cut • Enzymes that paste complementary DNA fragments together – DNA ligase

Using a restriction enzyme and DNA ligase to make recombinant DNA

Restriction fragment analysis by Southern blotting Characteristic pattern of bands for each sample DNA is transferred to paper and denature to single strands Entire genome Probe complementary to the DNA sequence of interest DNA bound to radioactive probe exposes film

DNA CLONING AND ITS APPLICATIONS • Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids. • Cloned genes are useful for making copies of a particular gene and producing a gene product.

RESTRICTION ENZYMES • Restriction enzymes are essentially molecular scissors that cut DNA at specific nucleotide sequences. • They originate from bacteria and function as a defense system against viral invasion. They “restrict” viral DNA.

Restriction Enzymes • Cut DNA at highly specific points • Recognize specific sequences – Four to seven bases – Each is unique • Consistent results

STICKY ENDS • Most restriction enzymes cut double stranded DNA in an asymmetrical fashion. • These cuts leave single stranded nucleotide overhangs that are competent to hydrogen bond. • These overhangs are called “sticky ends”.

AGAROSE GEL ELECTROPHORESIS • One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis. • This technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size.

Gel Electrophoresis • Separation of DNA fragments • Based on size

Cathode Power source Mixture of DNA molecules of different sizes Shorter molecules Gel Glass plates Anode Longer molecules

Different Endonucleases Yield Different Patterns Taq 1 + Ava. II Taq 1 + Pst 1 E coli clinical isolates

Questions • 1. What is a nuclease? • 2. How does an endonuclease differ from an exonuclease? • 3. What is a restriction endonucleases? Write names of some restriction endouclease.

Questions • 1. What is a nuclease? • DNA held by covalent bond joining adjacent nucleotides in DNA is called a phosphodiester bond. The phosphodiester bond between nucleotide in DNA molecules are very stable unless they are physically stretched or exposed to enzymes name nucleases. • Enzyme capable of breaking (hydrolyzing) phosphodiester bonds in DNA molecules and classified into exonuclease and endonuclease. • 2. How does an endonuclease differ from an exonuclease? • Endonuclease digest DNA by breaking phosphodiester bonds in the interior of DNA molecule. Exonuclease enzyme digest nucleotides from the ends of the DNA molecule. • 3. What is a restriction endonucleases? Write names of some restriction endouclease. • Restriction endonucleases are a special class of Endonuclease from bacteria to cut DNA. Eco. RI & Hind III. These are enzymes digest DNA by recognizing specific short sequences of bases called palindromic.

Questions • 4. What are 2 restriction endonuclease (RE) that we used in our lab? Write DNA sequences these RE recognize. Do they produce sticky ends or blunt ends when they cut the DNA molecules? • 5. How does the number of restriction sites relate to the number of fragments produced for linear DNA or circular DNA? • 6. What is palindromic DNA sequence?

Questions • 4. What are 2 restriction endonuclease (RE) that we used in our lab? Write DNA sequences these RE recognize. Do they produce sticky ends or blunt ends when they cut the DNA molecules? • Eco. RI & Hind III. Both produce sticky ends when cut. • 5. How does the number of restriction sites relate to the number of fragments produced for linear DNA or circular DNA? • Eukaryotic DNA, always going to have one more or one less fragment than you have restriction sites. • Prokaryotic DNA, the number of fragments will equal the number of restriction sites. • 6. What is palindromic DNA sequence? • Reading from the same thing in both direction to read the sequences bases that restriction endouclease recognizes. For example, M’adam I’m adam.

Questions • 7. What is electrophoresis? What does agarose gel electrophoresis allow us to do? • 8. What is the chemical nature of agarose? • 9. What factors effect the migration rate of DNA through an agarose gel? • 10. For DNA molecules of equal sizes, how do the different shapes (conformation) of DNA differ in terms of distance traveled through an agarose gel?

Questions • 7. What is electrophoresis? What does agarose gel electrophoresis allow us to do? • It’s a gel that allows move fragment of DNA across by attracting DNA, which is negative (anode) to opposite side (cathode) positive side base on size, and conformation of DNA. It will migrate with current. • 8. What is the chemical nature of agarose? Polysacchirde & sea weed. • 9. What factors effect the migration rate of DNA through an agarose gel? Size, shape (conformation), and charge. • 10. For DNA molecules of equal sizes, how do the different shapes (conformation) of DNA differ in terms of distance traveled through an agarose gel? Supercoil travels the fastest, follow by linear, & nicked

Questions • 11. In your p. AMP electrophoresis experiment, why did you run a DNA ladder (lane 5) and undigested p. AMP DNA (lane 4)? • 12. Write some practical applications for use of restriction end nuclease?

Questions • 11. In your p. AMP electrophoresis experiment, why did you run a DNA ladder (lane 5) and undigested p. AMP DNA (lane 4)? • Lane 4 is control of DNA to see what uncut plasmid looks like. • Lane 5 is DNA ladder: Containing known base pair lengths and use to compare with fragments in lanes 1 -3. • 12. Write some practical applications for use of restriction end nuclease? • LOOK AT SLIDES 85 -107 on this presentation.

Applications of DNA Technology • Diagnosis of disease – Viral genome detection (HIV) – Genetic disorders (screen for defective genes – hemophilia, CF, breast cancer) • Production of pharmaceutical products – Insulin for diabetes • Gene Therapy – Replace or supplement of a defective gene DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products. An example of DNA technology is the microarray, a measurement of gene expression of thousands of different genes.

Manipulation of DNA • Selective breeding Cloning of DNA -Restriction endonucleases -Vector -Gel electrophoresis -PCR – Domesticated animals – Dogs – Corn • Molecular Approaches Uses of DNA technology – Power, precision and speed – Transfer of one gene – Transfer between species -GMO -Human Disease -DNA Fingerprinting -Bioremediation -Biological methods dealing with pollution, oil spills, pesticide residues. -Gene responsible for breakup of harmful products (enzyme) cloned into bacteria. -Bacteria are seeded into a contaminated area.

Other applications… • Environmental Uses – Mining minerals – Detoxifying wastes (oil, sewage, pollution) • Agricultural Uses – Transgenic organisms • Sheep with better wool • Pig with leaner meat – Genetic engineering in plants • Resistant to disease and spoilage • Delayed ripening • Forensic Investigation – Identifying criminal by DNA fingerprinting – Paternity tests

Therapeutic Cloning n Therapeutic Cloning ¨ Creates embryonic stem cells ¨ Produces material for organ transplants ¨ Has been challenged on ethical grounds

Reproductive Cloning n Reproductive Cloning Creates living child ¨ Produces offspring identical to parents ¨ Has been done in animals, not people ¨

Gene Therapy

DNA Fingerprinting • Identifies individuals – Disease prevalence – Forensics – Paternity • RFLP analysis • PCR amplification

Sickle Cell RFLP

Applications: Detecting mutations Detection of Sickle-Cell 94

RFLP – Restriction Fragment Length Polymorphism • • DNA cut with Restriction Enzyme Gel electrophoresis DNA hybridization Compare bands • Applications: Catching the bad guys • DNA fingerprinting • • -Cut DNA with Restriction Enzymes • -Gel electrophoresis • -Compare bands

Figure 20. 17 DNA fingerprints from a murder case PCR amplify small amounts of DNA from crime scene Digest DNA and compare pattern of bands – DNA fingerprint

MEDICAL APPLICATIONS • One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases. • We don’t really understand a genetic disease until we know the mutation, how the gene works, and how the protein product functions both normally and in the disease state.

HUMAN GENE THERAPY • Gene therapy is the alteration of an afflicted individual’s genes. • Gene therapy holds great potential for treating disorders traceable to a single defective gene. • Vectors are used for delivery of genes into cells. • Gene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations.

PHARMACEUTICAL PRODUCTS • Some pharmaceutical applications of DNA biotechnology: – Large-scale production of human hormones and other proteins with therapeutic uses – Production of safer vaccines

SOME EXAMPLES OF BIOTECHNOLOGY PRODUCTS 1. Tissue Plasminogen Activator- dissolves bloodclots. 2. Human growth hormone. 3. Insulin 4. Blood clotting factor VIII. 5. Recombinant vaccines such as for Hepatitis B. 6. Bovine Growth Hormone. 7. Tissue Growth Factor beta. 8. Platelet Derived Growth Factor.

FORENSIC EVIDENCE • DNA “fingerprints” obtained by analysis of tissue or body fluids can provide evidence in criminal and paternity cases. • A DNA fingerprint is a specific pattern of bands of RFLP markers on a gel. • The probability that two people who are not identical twins have the same DNA fingerprint is very small. • Exact probability depends on the number of markers and their frequency in the population.

SOME UNUSUAL PLACES FORENSIC SCIENTISTS LOOK FOR DNA EVIDENCE.

DNA FINGERPRINTS CAN BE USED TO DETERMINE PATERNITY

ENVIRONMENTAL CLEANUP • Genetic engineering can be used to modify the metabolism of microorganisms. • Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials.

AGRICULTURAL APPLICATIONS • DNA technology is being used to improve agricultural productivity and food quality.

ANIMAL HUSBANDRY AND “PHARM” ANIMALS • Transgenic organisms are made by introducing genes from one species into the genome of another organism. • Transgenic animals may be created to exploit the attributes of new genes (such as genes for faster growth or larger muscles). • Other transgenic organisms are pharmaceutical “factories, ” producers of large amounts of otherwise rare substances for medical use.

GENETIC ENGINEERING IN PLANTS • Agricultural scientists have endowed a number of crop plants with genes for desirable traits. • Herbicide resistance. • Resistance to pests and disease. • Improved nutrition.

GOLDEN RICE • Genetically modified to accumulate beta carotene (vitamin A). • Over a million children a year go blind from vitamin A deficiency.