Agarose Gel Electrophoresis
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- Опубликовано: 24 июл 2024
- Please note my mistake in describing the anode as negatively charged (and the cathode as positive). The opposite is correct: the cathode is negative; the anode is positive. This doesn't impact the accuracy of the description, though: the positive charge is placed at the end of the gel away from where the DNA is loaded, so that the negatively charged DNA migrates through the gel toward the positive terminal.
00:00 Introduction
02:35 The agarose gel
03:44 Electrophoresis
05:33 Analysis of samples
06:45 Staining DNA so that we can see it in the gel
07:39 Example results
08:06 Interpretation of the water (control) reaction
08:56 Molecular weight ladder and identification of band sizes
09:51 Questions
Questions:
1) What are the sizes of samples A-M?
2) Why are there differences between the children and the parents?
Answers to the questions at the end of the video:
1) What are the sizes of PCR products in DNA samples A-C (three children), P (paternal), and M (maternal)? Compare the positions (vertically) on the agarose gel to the band positions of the MW (molecular weight ladder) sample. As described in the video, there are four MW bands: 1,000 bp (base pairs), 500 bp, 250 bp and 100 bp. After gel electrophoresis, the smallest MW fragment will have moved the farthest down the gel (from top to bottom). So, from top to bottom, the MW bands are 1,000 bp, then 500 bp below it, then 250 bp below it, then 100 bp at the bottom of the gel. Individual A has a PCR product the same size as the 500 bp band (so that product is 500 bp long). Individual B has a PCR product the same size as the 250 bp band (so that product is 250 bp long). Individual C has two PCR products: one 500 bp and one 250 bp. Each of the parent DNA samples (P and M) produced two PCR products, just like individual C: one 500 bp and one 250 bp.
2) Why are there differences in PCR product band pattern (i.e. one large, one small, or one of each) between the parents and the children? In this example, the most likely explanation is heterozygosity. Many species (including humans, which I based this example on) are diploid: each person has two copies of each chromosome: one copy ("homolog") inherited from their father at fertilization; the other homolog inherited from the mother. Homologs are the same chromosome (e.g. human chromosome 6), which means that the paternal and maternal homologs have the same genes. However, homologs do have sometimes major and sometimes minor genetic differences between them. This is because of random processes like point mutation and generation of DNA insertions or deletions on chromosomes. So, most individuals are heterozygous at most chromosomal loci. Normally we teach heterozygosity using upper- and lower-case letters: at one locus (nucleotide) that has experienced a point mutation, for example, a male could be homozygous C (written C/C) and a female could be homozygous G (G/G) at the same spot on the same chromosome. When they have children together, that child would inherit a C from the father and a G from the mother, and thus be heterozygous (C/G). The same is true for insertion/deletion events. A father might have GAGACCCCCCCCCCTATA (GAGA, then ten C, then TATA) on one homolog, and GAGACCCCCCTATA (GAGA, then 6 C, then TATA) at the same locus on his other homolog. We'll call these the C10 and C6 alleles. Say that a father is heterozygous (C10/C6) and we PCR amplify his DNA at this locus using primers complementary to the GAGA and TATA sequences that flank the stretch of Cs. That PCR would create a mixture of two different products: one 18 bp long (from the "long" homolog) and one 14 bp long (from the "short" homolog). When that sample is analyzed on a gel, we will see a larger band and a smaller band. So, in the example from the video, it appears that the mother and the father are both heterozygous in this way: each has one "large" and one "small" stretch of nucleotides in between the primer pairs used in the video example. The final question, then, is: why do the children vary in their electrophoresis results? The answer has to do with Mendelian genetics and the concept of random fertilization. The father will create some sperm that have his C6 (small band) allele, and some that have his C10 (large band) allele. The mother will create some eggs that have her C6 allele and some that have the C10 allele. At fertilization, then, sometimes C6/C6 children will be created, sometimes C6/C10, and sometimes C10/C10. In the video, child A is an example of inheriting "large" (C10) alleles from each parent; child B is an example of inheriting "small" (C6) alleles from each parent; like the parents did themselves when they were created, child C has inherited one "small" and one "large" allele, creating another heterozygote (C6/C10).
Wonderful explanation !! Made it really easy for self-understanding as well as explaining someone with ease.
Thank you so much.
Thank you so much for uploading these, i’ve learnt more from watching your videos than half a year of highschool Biology. Cheers from down under!
Can you answer the questions ones again? Cannot find the answer..
thank you very much Dr Joseph (cathode -) and ( anode +) thank you again Dr. Joseph
Absolutely right - my mistake! Cathode is negative and anode is positive.
@@JosephRoss Thank you Thank you Dr. Joseph I apologize to you and very grateful to you
That was great I could watch this video all day
very useful videos ! thank you very much. But how can i check my answers for your questions that you put at the end of each video ?
Unfortunately, there is no easy way at this point, unless you are a student in my in-person class (in which case we discuss the answers in person during class).
Thank you - very useful. I missed the point that we generate millions of copies that are always shorter than the original template... -- implications of this are ???
That is a good question, which I normally discuss in my face-to-face classes, but I should have provided that background in the introduction to this video as well. There are many reasons for scientists to use the PCR technique to generate those millions of copies. One value of having a homogenous sample of identical DNA fragments that are small (for PCR, typically on the order of a few hundred to a few thousand base pairs long) is that they all migrate at the same rate through the agarose gel, so they form a band instead of a blurry smear on the gel. One way this technique is used is DNA fingerprinting, which is a forensic technique that can uniquely identify DNA samples based on a pattern of PCR band sizes from multiple loci on different chromosomes. It is only possible to accurately determine the size of each PCR product if they are all the same length and form a band on a gel. That's just one example. PCR is also critical for many forms of molecular cloning, like making recombinant DNA.
Joseph Ross Many, many thanks Joseph - so benefit are millions of identical fragments - that they are shorter than the original sample is irrelevant. Outstanding lectures - in content and form !!
Just hit me you draw everything backwards. Anyway, these are helpful, thanks.
No he draws them normally, just like you would write a sentance down on paper but he uses a computer software to invert the image, like in a mirror, so that the writing looks correct to us.