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Welcome to genetics world ..
the music of life.. in the key of DNA!
Haneen sba'ane &zainah matani .
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We will continue talking about physical properties
of DNA, last time we talked aboutHYPERCHROMICTY >>look at this figure:
A curve represents Hyperchromicity phenomenon of
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nucleic acids under different physical conditions:*the Y axis represents the absorbance of light at 260 nm
wavelength. The X axis represents the wave length (nm.(*these are measurements of the absorbance of nucleic acids
under different wavelengths "as you see in the figure" inDNAsolution.
*notice that this nucleic acid absorbs maximally at260nm.And this is a constant physical property of nucleic acids. " you
can conclude this number (260 nm) from the experiment"*if you study this curve carefully you will see that it represents a
spectra of nucleic acid which is single-stranded -the one above-"look again at the curve" , while the other one is spectrum ofnucleic acid of the double-stranded DNA.
*single-stranded DNA absorbs light more than double-strandedDNA
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DNA melting curve, Hyperchromicity here is usedto follow the effect of increasing temperature on
denaturation pattern of DNA: *melting DNA means : denaturation of double-stranded intosingle-stranded DNA.
*Y axis : percent of hyperchromicity ,, X axis : temperature.
*First we have a sample "or preparation" of DNA, westarted to measure the absorbance at 260 nm as we know.But here during measuring absorbance at constant
wavelength "260, we are increasing temperature >> noticethat increasing temperature will increase absorbance, untilwe have 100% hyperchromicity at 90 C.
*100%hyperchromicity means that DNA is fully denatured.*when hyperchromicity is 50% , the temperature then is
called "melting temperature" or Tm and at this point(midpoint), 50% of DNA is denatured, here we have 50%transition from "hypo to hyper" chromicity.
*every DNA molecule has its own Tm >> it depends on thecomposition of that DNA molecule, if rich in G+C ,Tm will behigh, if rich in A+T then it will be less.
*Tm is very important for diagnostic purposes.
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Hyperchromicity can be used tofollow the denaturation of DNA as a
function of increasing temperature.As the temperature of a DNAsolution gradually rises above 50degrees C, the A-T regions will meltfirst giving rise to an increase in the
UV light absorbance.As the temperature increasesfurther, more of the DNA willbecome single-stranded, furtherincreasing the UV absorbance, untilthe DNA is fully denatured above 90degrees C.
The temperature at the mid-point ofthe melting curve is termed"melting temperature" and isabbreviated Tm. The Tm for a DNAdepends on its average G+Ccontent: the higher the G+C
content, the higher the Tm.Note: G+C content, G-C content,
and GC content are equivalentterms.
We said that Tm is very
important for diagnostic
purposes. All gene
testing depend on
what's called PCR!!
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When a solution of double-stranded DNA is placed in a spectrophotometer
cuvette "an instrument to measure light intensity " 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).
If the same DNA solution is melted (denatured), the absorbance at 260 nm
increases to approximately 40%. This property is termed "hyperchromicity."The hyperchromic shift is due to the fact that unstacked bases absorb more
si tahW?
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This curve is thesame like theprevious one,but here wehave differenttypes of DNA
molecules:*We have
different Tmvalues for thesedifferentmolecules, all at
the same hyperchromicity percent "50.%*We said that Tm depends on its average of G+C content.
ALSO, it depends on the ionic strength of the solution.*At fixed ionic strength there is a linear relation between Tm
and G+C content.*the curve at the left is for the DNA with the lowest amount
of G+C content ...(lowest Tm.(So .. we have proportional relation between Tm and G+Ccontent of DNA.
*Under the conditions used in this experiment, E. coli DNAwhich has an average G+C content of about 50%, meltedwith a Tm of 69 degrees C.
The previous pictures were to explain
denaturation of nucleic acid.
*Denaturation is important in:
-Genes expression.
-DNA replication.
-For diagnostic purposes in vitro.
*Factors that cause denaturation:
-Extremities of PH.
-Temperature.
What's the PCR?
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-Organic solvents.
if it's so big then reassociation "or rewinding" will takelong time.
NOW , if we have a repetitive sequence in the DNA molecule thenreassociation will take place faster than having non-repetitivesequence "also non-repetitive sequence is called one-single copy of
gene."we mean by one-single copy of gene thatthe gene is found only
once in that big DNA molecule . "presented in one copy per haploidgenome"
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>>Renaturation steps
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A curve showing the kinetics of reassociation of a DNAmolecule, we are not concerned with the details here,this curve is called "Cot curve for human genomic DNA
" shows a DNA Renaturation (reassociation) reaction:*The reaction follows ideal second-order kinetics. "Cot" is
the product of Co (initial DNA concentration) and t (time(*This curve is composed of three regions "three profiles
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Human genome consist of these types of DNA:-Slow fraction "single copy: it makes 75% of our
genome
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*
E
X
A
M
P
L
E
S
o
f Interspersed Repetitive sequences:
-ALU sequence: they are sequences of
deoxynucleotides , about 300 base pairs
(BP) in length , they have specific sequence and they are
repeated 300000 times in the genome , they could be
everywhere "in front of the gene, at the end of the gene,within the gene INTERSPERSED". As we said they are
found every where, not only in the genes!! We should
know that our genes represent 1.5% of our genome and
the rest represents DNA that does not code for any
protein "is not considered as gene. SO Alu sequence could
be found every where in DNA .Their presence can
sometimes leads to the occasional disruption of genes.
-VNTR "variable number of tandem repeats" : short repeated
sequences of only a few base pairs , variable in length ,
they interspersed throughout the genome.
They are highly polymorphic "differ in length or number of
repeats from individual to individual" SO they are useful
for mapping genes.
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*The importance of these repeats:
>This is important to test paternity
problems. NOW concerning Identical
twins they have the SAME DNA
fingerprint, but non-identical twins have
different DNA fingerprints
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You may read in your BOOK about what is called "satellite..."
*First "as you know", the satellite repetitive of
DNA sequence has the same meaning ofTandem
repetitive of DNA sequence.
*We have the term minisatellite: number of
nucleotide in that repeat is between 10_60
nucleotides.
*Microsatellite: number of deoxynucleotides
below 5.
>>Also you could see dinucleotides repetitive
sequences
"e.g. GCGCGC" 100000 of times within your
genome.
>>Also you could see trinucleotide repetitive
sequence in all over your genome.
ALL these repetitive sequences have significance in
diagnosis identification and linkage to disease as
you would see through the coarse.
Knowing the complete sequence of the human
genome will allow medical researchers to more
easily find disease-causing genes. In addition, it
should become possible to understand how
differences in our DNA sequences from individual
to individual may affect our predisposition to
diseases and our ability to metabolize drugs.
Because the human genome has ~3 billion bp of
DNA and there are 23 pairs of chromosomes in
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>
*The Gene contains:
- Promoter region.
- Exons.
- Introns.
- 3' & 5' regions.
chromosome has ~130 million bp DNA.
*A picture to show you the
complexity and size of
genomes in different
organisms.
*the human genome projectwas finished at 2000.
*it was thought that the
number of human genome
was between
(80000_140000) genes, but
after they did all the
sequences of human
genome they discovered
that the total number of our
genes ranges between(30000_40000) genes or
Let's take a piece of
chromosome, eachchromosome has a single
double stranded DNA
molecule , specifically
this is a picture shows a
sample of a gene within
the double stranded DNA
molecule on the
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NOW, let's talk about gene structure in
details:
1) Promoter region: it's a specific
DNA sequence that contains
regulatory sequences, they will
not code for any protein>> JUST
regulatory sequences.
These regulatory sequences are important to be
recognized by specific proteins, to initiate the DNAreplication or transcription or any other activity of the
gene.
USUALLY, promoters are found in the 5' region of any
gene.
"May be found within the gene OR in 3' region too"
>> the first nucleotide after promoter region will take
the number +1, it's found at the 5' end of the first exon.
The nucleotides which are before it are going to take
the minus numbers.
In order to assign for those regulatory sequences in the
promoter region, one will identify to say for example:the sequence at -25 regions are important in this
promoter for this gene etc.
2) Exons: they are sequences that code for proteins
when they are transcribed and translated into amino
acids.
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3) Introns: between Exons, they are DNA sequences in
any gene that don't code for any protein.
>> Most genes in the human genome are called "split
genes" because they are composed of "exons" separated
by "introns."
*SOME NOTES :>> The transcribed region of a gene (double-endedarrow :The arrows here are stop signals meaning , just to
show you the boundaries and it is the ends) starts at the +1
nucleotide at the 5' end of the first exon and
includes all of the exons and introns. (Initiation of
transcription is regulated by the promoter region of
a gene, which is upstream of the +1 site). To givewhat's called immature mRNA or immature RNA.
>> Not only mRNA will be transcribed, there are
genes for tRNA (transfer RNA) and rRNA (ribosomal
RNA) that also will be transcribed.
>>we have a region in the Promoter region called5' flanking region, and the region at the end of the
gene that doesn't contain any exons or introns is
called 3' flanking region, they could have some
regulatory sequences also.. (it will not be transcribed
nor translated)
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SO >> 3' flanking region, and 5' flanking region will not
be transcribed nor translated!!!
>> The 5' region of exon number one will not be
translated, it will just be transcribed. Also somesequences of them are in order to identify the
beginning of translation. Look at the figure again ...
look at the 3' & 5' regions in the transcribed RNA, these
light in color regions are not translated to proteins"
>>Primary RNA (could be mRNA, tRNA, rRNA ... all
called primary transcript) that resulted fromtranscription, will be processed by removing of
introns and connecting of exons by specific
proteins before being translated. This process
takes place in
the nucleus.
?What's the
function of
introns!?
You know that
the whole
genome size is
3 billion base
pair, now...
Only 1.5 % of
these base
pairs are
GENES.
What is the
function of the rest 98.5%? In another meaning what's
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the function of introns?!!1(Some of them are for regulation of gene expression; some
of those sequences will participate in the regulation.
2(Others are for protection of our genes, as you know ourgenome is always exposed to external factors and mutations,
and the presence of these extra pairs will protect our genes
from destruction from those external factors, so the presence
of this huge extra portion in DNA will protect it from mutations.
?What are those empty boxes? In mRNA.. Figure p. 13Those empty boxes are part of the exons that are not
translated ,you know that at the beginning of translation, the
transfer RNA recognize for the methionine codon to start the
translation process .We have many codons before the transfer
RNA sees methionine codon ,so all these codons will not be
used for translation ,there could be 20 or 30 or 40 codons ,but
the transfer RNA sees the methionine codon and begins the
translation process from there , so this region (the emptyboxes ) could undergo transcription but
can't undergo translation because it
doesn't have to this point the
methionine codon.
?Why arent there exons in the 3' flanking
region?
Because the gene ended,
every gene is composed
of a specific number of
exons ,there is no fixed
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number of exons for all the genes ,so the genes are
variable in size and in the number of exons .The 3'
flanking region is a noncoding region ,they will not code
for any proteins or any amino acids ,here we have a signal
of DNA sequence in the gene that will tell the protein that
this is the end of this gene and another gene will start , so
these introns or flanking regions will make boundaries
between genes within the genome.
look at this figure :
* this figure shows example of the wide variety of genestructures seen in the human genome .
*some genes do not have introns, example is histone
genes. They have one exon , they function in synthesizing
histones. Histones are: Basic proteins that are important instabilizing the double helical structure of DNA.
*Beta-globin gene: is the gene that is responsible for
synthesizing the beta globin polypeptide chain which is thesubunit of hemoglobin.
*HGPRT: hypoxanthine guanine phosphoribosyl transferase,
has exons.
*Factor XIII: which is a clotting factor, has exons.
And here's also a gene for dystrophin which is very important
in muscle contraction and function, this is the biggest gene inour cells (dystrophin ) that has hundreds of exons.
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minus.. the same ,while introns in genes are different in
sizes in genes.
SO .. VARIATION IN GENE SIZES IS DUE TO VARIATION IN
INTRON SIZES AND NOT EXON SIZES>> THE INTRON
DETERMINES THE SIZE OF THE GENE.
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This curve represents 3 columns, first column represents
cholesterol concentration in blood in normal people ranging
from ( 100 to 220 mg cholesterol /100 ml blood) , second one
represents the concentration of cholesterol in people who are
heterozygous for familial hypercholesterolemia the
accumulation ranging between ( 300 to 500 mg cholesterol /
100 ml blood) which is higher than normal ,while people whoare homozygous for the disease have the highest
accumulation of cholesterol with a concentration ranging from
(700 to 1000 mg cholesterol /100 ml blood) and next lecture
I'll explain it on the molecular level.
_____________________________________________________________^
^____
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- The happy end _^ , I'm happy of this end aktar
wa7de feekom :P
Bidayatan b3rf enu el mo7adara 6weele $wai ! w ma b3rf
$o el fikra elli 3ind el 6olab lamma tkon el mo7adara6weele bi9eero yid3o 3la elli fara3'ha :P but really it's not
my fault :P
>>mo3zam el slides elli en$ara7 3leehom are included
here ,every thing mentioned also is included here , I did
also include some notes of al 8o9oor lectures "la tawdee7
el 2$ya2 elli kan bidha tawdee7 "
Special thanx from me to zainah matani > it's not being normal that'simportant , but learning to accept our being different : to
live and love as fully as we can ;)
w el ehda2 la kol 9a7bati , w kol el $okor wl ta8deer lal
nas elli kanat wa2fe m3i and giving support thanx !
DONE BY :
HANEEN SABA3NE
ZAINAH MATANI ..