Moayyad Al-shafei. Mohammad Tarabeih. Dr Ma'mon Ahram. 1 P a g e

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1 3 Moayyad Al-shafei Mohammad Tarabeih Dr Ma'mon Ahram 1 P a g e

2 In this sheet, we are going to discuss 2 main topics: 1- The advantages of restriction endonucleases. 2- DNA replication. Before we start our lecture, let s revise some useful points which may help us to understand today s lecture: -In the gel electrophoresis: DNA molecules are separated based on size (length/molecular weight). Then the DNA is stained, and the DNA molecules will appear as bands. -In the Hybridization techniques: Hybridization reactions are used to detect specific nucleotide sequences. This technique uses a probe. -Southern blotting technique: This technique is a combination of DNA gel electrophoresis and hybridization so used to detect: A) The presence of a DNA segment complementary to the probe B) The size of the DNA fragment. -Endonucleases: Enzymes that degrade DNA within the molecule rather than from either end (exonucleases). -Restriction endonucleases: Enzymes that recognize and cut (break) the phosphodiester bond between nucleotides at specific sequences (4- to 8-bp restriction sites) generating restriction fragments. Examples of restriction endonucleases: EcoR1 (isolated from E. coli): recognizes GAATTC and makes a cut within this sequence (between the G an A nucleotides). Hinf1 (from Haemophilus influenzae): recognizes ANTC ('N' is any nucleotide). So it cuts at AATC / ATTC / AGTC / ACTC. 2 P a g e

3 NOW let s start our discussion: restriction endonucleases can be used in many techniques but we will discuss only 2 of them which are: 1- Restriction fragment length polymorphism (RFLP) 2- Cloning Before talking about RFLP, we will have a brief introduction about the DNA polymorphisms: Poly means many and morph means shape, so the term DNA polymorphisms means different shapes of DNA. Remember that we are diploids; we have a maternal and paternal copy of each chromosome. The similarity among individuals is 99.99%, so there is 0.01% variation which makes the differences among us. What makes the variation is the replacement of one nucleotide at a certain location on the DNA by another. These variations can be significant or nonsignificant. An allele: is a gene type that encodes a certain feature; if the variation takes place at this location of the DNA, different features will appear. EX: All of us have the eye color gene, different types of this gene are called alleles. Each individual has a different allele that determines a different colour of the eye; so, one allele makes them black, the other makes them blue and etc. Alleles can be Homozygous if they are exactly the same or Heterozygous if they are different. What is the effect of the DNA polymorphisms on the restriction digestion? See the figure below, suppose that we have 2 alleles: Allele 1 on the left, by using the EcoR1,3 restriction sites will be recognized by the enzyme generating 4 fragments. Allele 2 on the right with the same DNA sequence except that it has a polymorphism by changing one nucleotide at the restriction site (GAATTC); making it unrecognizable to EcoR1 enzyme. So, by using the EcoR1, 2 restriction sites will be recognised by the enzyme generating 3 fragments only which 3 P a g e

4 have different lengths than the fragments without polymorphism. -From this we can understand that the RFLP technique means the presence of DNA polymorphism in individuals generating differences in the length of the restriction fragments We can detect the RFLP by using the Gel electrophoresis OR Southern blotting, but how?! let s have an illustrative example: See the figure below, assume that we are studying the variations in a certain gene in 3 cases and there are 2 variations(alleles): the first (variant 1) has GCATTC sequence which can t be cut by EcoR1 and the other (variant 2) has GAATTC sequence which can be cut by EcoR1(between the G&A). (remember that we are diploid, having 2 copies of each gene (Homozygous or Heterozygous)) Case 1: Let s say that there is a person who has the both copies of allele that has GCATTC (variant 1) sequence which can t be cut by EcoR1 in either chromosome, remaining as a long uncut fragment. And if we used gel electrophoresis they (the maternal and paternal uncut fragments) will move slowly as a one band. (So this person is homozygous for this location of the DNA). 4 P a g e

Case 2: Let s say that there is a person who has both copies of allele that has GAATTC(variant 2) sequence which can be cut by EcoR1 in both chromosomes generating two shorter fragments (for each

5 Case 2: Let s say that there is a person who has both copies of allele that has GAATTC(variant 2) sequence which can be cut by EcoR1 in both chromosomes generating two shorter fragments (for each allele). And then we used gel electrophoresis they will move faster. (so, this person is homozygous for this location of the DNA). Case 3: Let s say that there is a person who has one allele that has GAATTC(variant 2) sequence which can be cut by EcoR1 and the second allele that has GCATTC(variant 1) sequence which can t be cut by EcoR1. This form is heterozygous and this will generate a long fragment from the uncut allele (variant 1) and two shorter fragments from the second allele (variant 2) that has been cut. Note: So RFLP combined with gel electrophoresis can help us to determine if the person is homozygous or heterozygous. That was the use of gel electrophoresis with the RFLP, what about the use of the Southern blotting technique? Southern blotting > we will use a probe which will recognize a certain sequence of the DNA. See the figure below, assume that we have 2 DNA fragments: The first case at the left (molecule 1), there are 3 restriction sites for thee BamH1 (GGATCC) which will be cut into 4 fragments and our probe will hybridize to a short fragment 4 kb. While in the second case at the right (molecule 2), we have the same fragment except there is a mutation which changed one of the 3 restriction sites (GGATTCC to GGGTCC) making it unrecognizable to BamH1; generating only 3 fragments and our probe will hybridize to a long fragment 9kb (see the figure). 5 P a g e

6 If the person was homozygous for the GGATCC allele, the probe will detect the 4kb fragment only. And if he was homozygous for the GGGTCC allele, the probe will detect only the 9kb fragment. A person is heterozygous, has both GGATCC & GGGTCC alleles, we can expect that the probe will detect both 4kb (resulted from the GGATCC allele) and 9kb (resulted from the GGGTCC allele) fragments. *Notice that the fragment that has the probe when mutated is much longer than the fragment that had the probe with no mutation, so by gel electrophoresis we can determine the size of the fragment that has the probe. (The probe is used mainly if the amount of the available DNA is not sufficient to be viewed on the agarose gel by itself, so the probe amplifies it). Now guess that the probe detects a region that contains a restriction site: The rule here is As long as the probe can form enough hydrogen bonds with a DNA fragment, it hybridizes. If there is a cut, the probe will detect both fragments that surround the restriction site, but if there is no cut, the probe will detect only one fragment contains the mutated restriction site. Let s have an example to simplify these case: In the following figure, the probe detects a restriction site located between the red and green parts of the DNA. as usual we have 3 cases: Case # 1 (morph 1) at the left, when there are 3 restriction sites (one of them between the green and the red fragment) and the person is homozygous for this allele. So, the probe detects both the red and the green fragments separated. 6 P a g e

7 Case #2 (morph 2) at the right, there are 2 restriction sites (the one between the red and green parts is mutated) and the person is homozygous for this allele. So, the probe detects a long fragment that contains both the red and green parts. Case # 3 when the person is heterozygous we can expect that the probe will detect both the red and the green fragments separated (similar to morph 1) and a long fragment contains both the red and green parts (similar to morph 2). -RFLP technique as a diagnostic tool For example, if a mutation that results in the development of a disease also causes the generation of distinctive RFLP fragments, then we can tell: if the person is diseased as a result of this mutation from which parent this allele is inherited Sickle cell anemia caused by single nucleotide mutation, which changes the amino acid Glu into Val, in the globin gene; the location of the mutation has been determined and it has been found that the site of mutation is within a restriction site for an endonuclease called MstII which makes a cut only if the gene is not mutated. 7 P a g e

And by using a probe that covers this restriction site: The normal homozygous person AA will have two fragments (because of the cut) which are recognized by the probe.

If the person is carrier Aa e will have one normal allele and one mutated allele, so we will have one large fragment coming from the mutated allele and two short fragments coming from the normal

8 And by using a probe that covers this restriction site: The normal homozygous person AA will have two fragments (because of the cut) which are recognized by the probe. The person who has sickle cell anemia aa, homozygous for the mutated allele (the two alleles can t be cut by MST1) meaning that the probe will recognize a single large DNA fragment. If the person is carrier Aa e will have one normal allele and one mutated allele, so we will have one large fragment coming from the mutated allele and two short fragments coming from the normal allele. In order for an individual to have the disease, he must take the mutated copy from both parents. In the figure, here both parents are carriers (Aa) for the disease so electrophoresis shows 3 bands. In The diseased child (aa) we will see only one band of a large fragment. The normal child (AA) will have only two bands of smaller fragments The carrier child (Aa) will look exactly the same as parent. Paternity testing -Paternity testing is performed to determine the parents of a child. we collect DNA samples from the mother, the father and the child. -Every single DNA fragment in the child must come from one of his parents, but not equally acquired! that means the ratio of the fragments acquired from the father and the mother is not necessarily 1:1(it may be 1:1 but it depends on the genes tested and their location on the DNA). note: it is not a must that every chromosome found in the mother and father exist in the child. 8 P a g e

Let s take an example From the figure below: For D1: The first and last DNA fragments come from the mom and the 2nd and 3rd fragments come from the dad, so she is their daughter.

9 Let s take an example From the figure below: For D1: The first and last DNA fragments come from the mom and the 2nd and 3rd fragments come from the dad, so she is their daughter. For D2: there are DNA fragments in the daughter that match the DNA fragments in the mom, but not the dad, so she is not his daughter. For S1: has DNA fragments that look like the fragments of the father or the mother, so he is their son. For S2: DNA fragments are not similar to either of the parents, so he is not their son. (adopted) Forensics: -Another use of RFLP is in Forensics. In the crime scene, blood spots help to identify the murderer by doing DNA analysis (we compare possible suspects DNA with that in the crime scene) -A part these blood spots may have come from the victim and part may have come from the murderer (contamination by bacteria or DNA from the investigators themselves may occurs). - Now to know who is the real murderer, by using RFLP technique we generate a pattern for each of the suspects. -Then we compare the pattern from the DNA extracted from the blood found in the crime scene with the pattern of each suspect. 9 P a g e

- Murder's blood must be 100% present in the pattern of the blood we found in the crime scene. But the blood we found may contain extra fragments due to the mentioned contamination.

- By using RFLP technique we generate a pattern for this blood and by comparing the pattern from the DNA extracted from the blood found on the clothes with the pattern of the victim we can know who

10 - Murder's blood must be 100% present in the pattern of the blood we found in the crime scene. But the blood we found may contain extra fragments due to the mentioned contamination. To understand this process, see the figures below: - We may find some clothes in the suspect s house which are contaminated by blood (thought to be victim s blood). - By using RFLP technique we generate a pattern for this blood and by comparing the pattern from the DNA extracted from the blood found on the clothes with the pattern of the victim we can know who is the real murderer. Cloning Cloning is another way we could take advantage of restriction endonucleases. Cloning means that you make several copies of one thing A clone is a genetically identical population, whether of organisms, cells, viruses, or DNA molecules. Every member of the population is derived from a single cell, virus, or DNA molecule. How do we clone a DNA molecule? The DNA fragment of interest is inserted into a DNA carrier (called a vector) that can be replicated. An example of these vectors is the bacterial plasmid DNA. What we basically do is that we open a plasmid, insert the fragment of interest inside the plasmid, close it, and then transfer 10 P a g e

11 the plasmid into a bacterial cell that can replicate and make copies of this plasmid. This plasmid after we inserted the DNA fragment of interest is called a recombinant DNA (DNA molecule which is made from different sources). In order to clone a DNA fragment, a plasmid or any other vector must have three characteristics: 1- It must replicate independently of bacterial chromosomal DNA (bacterial cell may have multiple copies of plasmid while it has only one original copy of its chromosomal DNA). 2- Can insert a foreign DNA fragment 3- The plasmid must have a selectable marker (drug resistance gene) We can have an antibiotic resistance gene in the plasmid and if the bacteria contain the plasmid, the antibiotic won t affect it and it will survive. Killing all the other bacteria that don t contain this plasmid And giving a purer culture. Now let s talk about this process in details: 1- We cut the plasmid with an endonuclease (EcoR1 which cuts at GAATTC) opening it and generating a linear DNA molecule with sticky ends. (I already know that this plasmid contains a specific restriction site) 2- we do the same to the DNA fragment, which is already liner, in order to produce sticky ends 3- Because both the plasmid and the DNA fragment are cut with same restriction enzyme (thus have the same overhangs), the ends of the fragment can bind to the ends of the open plasmid (they are complimentary to each other) forming a recombinant DNA. note: it isn t fully stable need step 4 to stabilize (DNA ligase) 4- we add a DNA ligase to close the recombinant DNA via phosphodiester bond. 5- We add an antibiotic to kill all the bacteria without this plasmid and only the bacteria with plasmid will survive. 6- The survived bacteria will multiply generating 11 P a g e

12 billions of copies in a short time with a lot of plasmids inside them 7- We extract this recombinant DNA (plasmid) from the bacteria. Then to get the DNA insert we use the same endonuclease enzyme used previously (EcoR1) which cuts at the same restriction site and frees the insert. DNA replication a general mechanism: In order for life to continue, DNA must be replicated. Some basic information (found in the slides): The entire DNA content of the cell is known as genome DNA is organized into chromosomes. Bacterial genome: usually one and circular chromosome. Eukaryotic genome: multiple, linear chromosomes complexed with proteins known as histones. How the DNA is replicated? -A parent cell has dsdna and each of its two daughter cells have an exact copy of the parent dsdna. -Different suggestions on the possible mode of DNA replication: 1- Conservative model: When the parental DNA is replicated, then the two DNA old strands will go to one daughter cell and the new strands will go to the other daughter cell. And that s wrong as each cell's DNA was proved to have pieces from the old strand and pieces from the new strand. 12 P a g e

13 2- Semiconservative model: When parental DNA is replicated, each daughter cell will end up with one old strand and one new strand, this is what actually occurs in our DNA. 3- Dispersive model: A random process in which the daughter cells have DNA strands that are a mixture of old fragments and new fragments. Later experiments were done to determine the direction of replication and they found that DNA replication occurs Bidirectional (in two opposite directions). But we know that the new nucleotides can only be added at the 3 end so the direction is from 5 to 3 always at the new strand. - When the DNA starts the replication process the first thing that must occur is the unwinding of the two double strands by a helicase enzyme forming what is called replication bubble.each half of the replication bubble is a Y-shaped active structure called a replication fork. Notice that we have two templates, one of them running from 3 to 5, and the other one running from 5 to 3 (Because the two strands of DNA are antiparallel). - The first template (3` to 5` at the original DNA) will make the new leading strand (from 5 to 3 ). -The second template (5`to3` at the original DNA) will make the new lagging strand (lagging means there is delay in synthesis and this delay occurs because the replication here occurs in short and discontinuous fragments those fragments known as okazaki fragments) -The leading strand is the responsible for the unwinding of the two strands and allowing the formation of the replication fork plus giving space to the lagging strand to replicate and that s why it is slower than the leading strand. 13 P a g e

- Components of DNA replication: DNA polymerases can t initiate replication de novo (from scratch).

For leading strand, we need only one primer, while for okazaki fragments we need a primer for each single fragment.

In the lagging strand, each fragment requires a primer and DNA elongation occurs by the enzyme DNA polymerase as usual.

14 - Components of DNA replication: DNA polymerases can t initiate replication de novo (from scratch). So, they require a RNA primer that is complementary to the DNA template to be added first. For leading strand, we need only one primer, while for okazaki fragments we need a primer for each single fragment. This primer is a short RNA sequence added by an enzyme called primase. Then the DNA Polymerase starts from the primer the process of DNA elongation by adding deoxyriboneucleotides. In the lagging strand, each fragment requires a primer and DNA elongation occurs by the enzyme DNA polymerase as usual. Now when the polymerase hits the primer of the following fragment it removes the primer (of the following fragment) and adds a deoxyriboneucleotides. When the polymerase finishes the synthesis of the Okazaki fragments they get connected to each other to form a strand by an enzyme called ligase. We said that For DNA synthesis to proceed, the DNA double helix must be opened up ahead of the replication fork Opening up the DNA is done by two types of protein contribute to this process 14 P a g e

15 1- DNA helicases: use ATP to open up the double helical DNA as they move along the strands. 2- single-strand DNA-binding proteins In bacteria, helicases form a complex with the primase called primosome Single-strand DNA-binding (SSB) proteins They bind tightly to exposed single-stranded DNA strands without covering the bases, which remain available for templating. They have a three functions:- 1. Preventing the formation of hairpin structure which happens when we have two complementary parts on the same single strand DNA they may bind together. If this thing happens the polymerase will be forced to stop replication. 2. Protect the single stranded DNA from the degradation by enzymes like DNase (as they are thought to be a viruses DNA) 3. Preventing the single strands from binding together again (rewinding). quick note: DNA synthesis continues from 5 to 3, the template is read from 3 to 5. U R BUILT TO CARRY ON 15 P a g e

Molecular Biology (2)

Molecular Biology (2) Restriction endonucleases, RFLP, and gene cloning Mamoun Ahram, PhD Second semester, 2017-2018 Resources This lecture Cooper, pp 120-124 Endonucleases Enzymes that degrade DNA within