_ DNA absorbs light at 260 wave length and it s a UV range so we cant see DNA, we can see DNA only by staining it.
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1 * GEL ELECTROPHORESIS : its a technique aim to separate DNA in agel based on size, in this technique we add a sample of DNA in a wells in the gel, then we turn on the electricity, the DNA will travel in the gel from () to (+) because of the () charge of DNA due to the phosphate group, se we have the smallest one ( low molecular weight ) in bottom and the largest one (high molecular weight ) in the top close to the wells. _ DNA absorbs light at 260 wave length and it s a UV range so we cant see DNA, we can see DNA only by staining it. _ the white color that we see in DNA is not its color, its the color of salt that bind DNA. _ to stain DNA we use adye called ethidium promide which is a chemical that integrate into DNA and give its orange color. * in picture in slide 58 the first line indicate asize standard which come from the company or we prepare it in our lab, it contain DNA fragment of known size ( 1000 base pair means it contains 2000 nucleotides ). _ in sample 1 & 2 the DNA appear as a band, each band contains thousands of DNA molecules of the same size. the first band of sample 1 & 2 have the same size because they travel to the same distance, the other bands travel to different site ; so we can differentiate between the tow sample.
2 * another way to detect DNA is by labeling it with radioactive phosphorus ; because DNA contain phosphorus and protein don t ; so we can differentiate DNA from protein. _ if we want to label protein but not DNA, then we use sulfur ; because sulfur present in protein but not in DNA. * HYBRIDIZATION TECHNIQUES : _ hybrid DNA : DNA fragment in which the 2 strand come from 2 different source. _ hybridization is a technique that involve renaturation of 2 strand of DNA from 2 different sources. *TYPES OF HYBRIDIZATION : 1 southern blotting. 2 northern blotting : hybridization techniques for RNA. 3 western blotting : hybridization techniques for protein.
3 for hybridization techniques and to detect DNA fragment we use something called PROBE (( a short DNA fragment of known sequence, that is radioactive labeled )) which added to the sample, if the PROBE binds to the long DNA fragment then the same sequence present. we may use this technique in DNA homology. * scientists have use yeast cell in experiment to understand mechanism that take place in human cell because it : 1_ simple eukaryotic. 2_ single cell organism. 3_ has 4 linear chromosomes ( not circular like bacteria ). 4_ easy and less complex than human cell. So if I find agene and I know its sequence, then i would to know if this gene present in human cell or not, and if it present, where? so I make a short PROBE based on yeast DNA sequence, and I label it, and add it to the human genome, if there is hybridization that means the gene exist in our body. * DNA is double strand so how the PROBE bind to it? 1_ by denaturation, OR 2_ add a large amount of the PROBE, it will compete with the long strand, the PROBE will win due to its large amount.
4 * southern blotting allow us to experiment 2 thing : 1 is the DNA segment complementary to the PROBE present or not. 2 the size of DNA fragment. *in southern blotting we cut the human genome into small fragment, then we separate the DNA fragment through agarose gel, and put it on a membrane to transfer DNA fragment from the gel to the membrane, then we add the radioactive PROBE ; the PROBE will bind the DNA fragment with the same sequence, (( from arrangement of DNA fragment on the membrane we can detect the size )). *RESTRICTION ENDONUCLEASE, RFLP, AND GENE CLONING : _endonuclease : are enzyme that cleave nucleic acid like proteases ( cleave protein ). _ esterase : cleave an ester bond. _ phosphatase :cleave or break phosphate group. _ exonuclease : cleave DNA at its end ( periphery ). _ restriction fragments: Enzymes that recognize and cut (break) the phosphodiester bond between nucleotides at specific sequences (4to 8bp restriction sites) generating restriction fragments
5 _ restriction fragments : fragments that are cut at specific site and have specific sequence. _ restriction sites : sites where the restriction endonuclease cut the DNA and they are usually 48 bp in number. _ these endonuclease are present in bacteria ; the immunological significant of these enzymes are to degrade foreign DNA ; those bacteria sometimes infected viruses and in order for these bacteria to protect themselves they synthesize endonuclease that cut the DNA of the virus or any foreign DNA. _ type II restriction endonuclease : which recognize the sequence and always cut at the same specific site e.g: EcoRI which an enzyme isolated from E.coli and recognize the sequence ( 5'GAATTC3' ). * in the exam be attention to the direction of the sequence (5' 3' ). _ in the EcoRI enzyme ; if we read the opposite sequence from 5' it will be GAATTC ; its a mirror image that called palindromic sequence and the half of the sequence is complementary to the other half. _ some enzyme are more free such as : HinFI which isolated from haemophilus influenzae and recognize the ANTC sequence ( where N is any nucleotides )
6 * Restriction enzymes cut DNA in two different ways: 1 Blunt : cut DNA like knife, at the same position on both strands, generating DNA fragments that are smooth ended 2 Staggered (offcenter) : Cut the two DNA strands at different positions, and they called sticky or cohesive ends because when the enzyme is terminated they recombined to each other by hydrogen bond ( not phosphodiester bond ) so they are less stable. * 5 vs. 3 overhangs : the free single strand DNA can end with 5 or 3 depend on the site of the cut. if the cut is close to the 5 then its 5 overhang, and if the cut is close to the 3 then its 3 overhang. *DNA ligase : an enzyme that ligate DNA fragment by forming 3 5 phosphodiester bonds between the 3hydroxyl end of one strand and the 5phosphate end of another strand, and it needs ATP for its function. *Advantage of restriction endonucleases :
7 1 Restriction fragment length polymorphism (RFLP) : generation of DNA fragment of different shapes by restriction endonucleases. 2 cloning. * As mentioned in the slides that the restriction endonucleases are used in RFLP which is simply defined as is a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples in question with specific restriction endonucleases." showing and detecting variations in homologous DNA sequences. " * We are diploid : which means that there are two copies for each chromosome except for the x and Y chromosomes in males which means that we have two copies for each gene so we've two alleles "types of gene coming from parents" for each gene, and as you know that a gene might be dominant on the other and every type of allele codes for different sequences "such as in the eye color genes" and as the two copies of the gene are coming from two different origins "parents" they might be similar "giving rise to a heterozygous genes" or different from each other " giving rise to a homozygous genes " * RFLP is detected "for example" by applying EcoR1 endonuclease on a DNA fragment as it detects the site of a GAATTC sequence and cuts the fragment at that site. by knowing this and comparing the results "number of fragments"
8 of applying EcoR1 on different alleles is going to show the variation between them. * Detection of RFLP : 1 Gel electrophoresis. 2 Southern blotting. *example in slide 81 : _on applying EcoR1 on variant 1 then along DNA fragment will appear in the gel because EcoR1 don t cut variant 1, so 2 fragment of the same type will be in the gel. _ on applying EcoR1 on variant 2 then a short 4 fragment of 2 types ( 2 bands ) will appear in the gel, because EcoR1 can cut variant 2. _ a person who has only variant 1 on his chromosomes OR only variant 2, then he is homozygous. _ a person who has both variant 1 & 2 on his chromosomes then he is heterozygous, and on applying EcoR1 then 3 bands will appear in the gel ; one long and tow short. * RFLP in the clinic : _ RFLP can be used as diagnostic tools in detecting mutation.
9 *example in slide 83 : _at first we add BamH1 to molecule 1 & 2, in molecule 1 the BamH1 will cut it into 2 fragment due to the presence of GGATCC sequence but in molecule 2 cut will not happen due to mutation of GGATCC to GGGTCC, then I separate them by gel electrophoresis, after that I transfer DNA fragment to a membrane then I add PROBE. the DNA fragments that will appear are a 4kp one and a 9kp one ( the one with mutation ). DONE BY : إحسان المعايطه
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