GENETICS ومن أحياها DNA Genes & Chromosomes الفريق الطبي االكاديمي DNA Genes & Chromosomes DONE BY : Buthaina Al-masaeed & Yousef Qandeel Page 0
T(0:44 min) In the pre lecture we take about the back bone of the double strand of DNA that are composed of phosphate group bound together by phosphodiester bonds,its covalent bond and in the center we have nitrogen bases that bond together by hydrogen bond. The two strand are complementary to each other that s mean if the C in one strand they must be G in other complementary strand of it,and if the A the must be T because the hydrogen bound between the complementary bases are the must stable and strong bound,if the HB between T and G it will be weak unstable bound and the whole structure will be destroy. C always with G,A with T very specific, T(3:58 min) Q from student >>what is the mean of 5to3 end in the double strand?? the bound that are form between subsequence nucleotides is bound by phosphodiester bound,so how phosphodiester form?? Its form between the 3 primary carbon atom of the sugar with the 5 primary of the next nucleotide. Always the phosphodiester bound that are form between nucleotide the are in form 3 primary to 5 primary. Each stander it has two end (begging and ending) The begging always characterize by having the 5 primary (free phosphate group ) while the end have free hydroxyl group. Page 1
T(7:11 min) The DNA it must be helix structure as we see and called (double strand helix structure) in this form of DNA will be function just in this structure (helically). Because of this twisted they will be major groove (large pocket) and the minor groove (small pocket),this groove is very important because in there process of any activation process of the gen like replication transcription or expression of the gene..there must be proteins to interact with the DNA. Those proteins must bounds very specifically at specific sit on DNA. Because of the major groove proteins can go to those major groove and according to the hydrogen bond there they could read the sequence of the nitrogen bases,if that sequence recognize by them in order to bond they will be binds,because the are very specific so for any protein to came and binds it must first scan the region by binding to the major groove and reading the sequence. The same thing for minor groove same proteins also will bind there but the bulk of proteins will bind to the major groove to affect the active process of the gens. Page 2
T(11:35 min ) There are 3 forms of DNA the most famous form is the B-form found in our cell, and also we have the A-form and Z-form. A-form was discover ( Rosalind Franklin). Why you have different forms of DNA??its according to sequence reference and also to the environment where the DNA is found. In most of the cases we have B-form and in genetic characterize which each term is composed of 10.5 bases per and its twisted right hand also A- form in right hand while Z-form in left hand ( opposite direction). As we see the Z-form is extended more than A-B form. In some cases of the dehydration A-form will be form. Z-form is important and act like a marker for regulation of gen expression, its mean when its gen near to expression the A-B form will be in Z-form. Z-form its zig zag form and its beating sequence of C and G. Forces affecting the stability of the DNA double helix hydrophobic interactions - stabilize - hydrophobic inside and hydrophilic outside stacking interactions - stabilize - relatively weak but additive van der Waals forces hydrogen bonding - stabilize - relatively weak but additive and facilitates stacking electrostatic interactions - destabilize - contributed primarily by the (negative) phosphates - affect intrastrand and interstrand interactions - repulsion can be neutralized with positive charges (e.g., positively charged Na + ions or proteins) T (15:49 min) Page 3
The doctor here took about the forces,this forces so important to stable the double helix structure ex hydrogen bond,. Phosphodiester electrostatic repulsion by negatively charged phosphates along the DNA backbone destabilize the double helix. hydrophobic interactions is between nonpolar in the ring of nitrogen bases. stacking interactions between the nitrogen bases. This slide shows base stacking and charge repulsion in the DNA double helix T(19:46min ) The forces stabilizing the DNA double helix can be overcome by heating the DNA in solution or by treating it with very high or very low ph (low ph will also damage the DNA, whereas high ph will simply separate the polynucleotide chains). When the strands of DNA separate, the DNA is said to be denatured (when high temperature is used to denature DNA, the DNA is said to be melted). Because some of the forces stabilizing the DNA double helix are contributed by base pairing interactions, and because A-T base pairs have only two hydrogen bonds in contrast to G-C base pairs which have three hydrogen bonds, regions of the DNA duplex that are A-T rich will denature first. Once denaturation has begun, there is a cooperative unwinding of the double helix that ultimately results in complete strand separation. Page 4
Now a day a lot of DNA sample use for genetic testing to diagnosed genetic disorder (take blood sample from the patient and repair it and we must take it away from high heat ). Denaturation process in vetro is very important for reaction called PCR its abbreviation for (polymerase chain reaction ) PCR in test tube you Denature the DNA into single strand and by specific reaction synthesis a billion of double strand DNA after Denature it also use primary to extend them according to single strand DNA and specific enzymes. DNA could be renature unlike proteins, if you remove heat, urea, PH so the DNA will renature or anneal to each other. Page 5
Electron micrograph of partially melted DNA This is how the DNA looks like under the electron microscope and this figure represent partial denaturation of the DNA. This happens due to base composition, regions with high A-T composition that has only 2 hydrogen bonds will "breath" first forming what is called "breathing bubble" but the C-G rich regions because they have 3 hydrogen bonds they will melt late. Hyperchromicity Page 6
Spectrophotometer: used to measure how much specific compound will absorb light & at which maximum absorbtion. When a solution of double-stranded DNA is placed in a spectrophotometer cuvette and the absorbance of the DNA is determined across the electromagnetic spectrum, it characteristically shows an absorbance maximum at 260 nm (in the UV region of the spectrum). Proteins absorb UV light maximally at 280 nm If the same DNA solution is melted, the absorbance at 260 nm increases approximately 40%. This property is termed "hyperchromicity phenomena". This is due to the fact that single-strand DNA is more exposed to UV light (more surface area exposed to the light) We can draw this curve depending on hyperchromacity at different temperatures. As you can see here hyperchromacity increase when we increase the temperature due to denaturation of DNA (melting) until we reach a point where there is no further increase in hyperchromacity because the DNA become single-stranded at this point. The temperature at the mid-point of the melting curve is called "melting temperature" or "transition temperature" ( Tm) and it is very important to be determined when you are performing PCR reactions Page 7
Each one of these curves represents a DNA from different species with different Tm due to difference in base composition (the more the DNA has C-G the more the Tm) Alu sequences are an example of interspersed DNA the function of them is not discovered but they will cause some genetic diseases and this will be explained later Page 8
Our genes and all mammalian genes are composed of introns and exons each gene has a 5' beginning that has free phosphate group and a 3' end that has free hydroxyl group Introns: are regions in the genes that does not encode for amino acids Exons : regions in the genes that code for amino acids Page 9