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BIOB11H3 Study Guide - Telomere, Globin, Hybridization Probe


Department
Biological Sciences
Course Code
BIOB11H3
Professor
Dan Riggs

Page:
of 6
Cell Biology Lecture 2 Paraniya Balakumar
Deoxyribonucleic acid (DNA) is a molecule encoding the genetic instructions used in the
development and functioning of all known living organisms and many viruses
DNA is made of a double stranded, antiparallel helix
Complementary base pairing occurs by hydrogen bonding in which A-T and C-G bond with
one another
Early work on DNA which uses the physiochemical approaches include:
1. DNA is a polynucleotide chain
2. Estimates made of genome size
3. Values were “tiny yet enormous”
Humans: 3.5 picograms of DNA/haploid
Genome: approxiamately 3.2 billion base pairs
4. Base pairing rules established by analyzing base composition
5. DNA absorbs light in ultraviolet range
6. Renaturation experiments defined complexity
Chargaff: (1950s) base composition differed between organisms
[A] = [T]
[G] = [C]
[A] + [T] doesn’t = [G] + [C]
o This is because AT bonds are held by 2 hydrogen bonds
o CG bonds are held by 3 hydrogen bonds
Absorbance measured by a spectrophotometer
It works by recording the ring structures of DNA which absorb in the ultraviolet range
DNA absorptions maximum range is approximately 260 nm
Absorbance can be used to determine DNA concentration
Absorbance increases about 1.5X if DNA is denatured. Thus changes in absorbance
reveal difference in % double stranded (ds) vs % single stranded (ss)
o This is because of the “satellite dish” analogy
o Single stranded DNA (ssDNA) is flexible and contains nitrogenous bases which can
freely rotate about glycosidic bond
o Double stranded DNA (dsDNA) is more rigid, nitrogenous bases are limited in the
number of positions they can occupy
Complexity is a measure of the number of unique (vs. repetitive) sequences that exist in a
genome.
There are lots of repetitive DNA in genomes
DNA reannealig (and later use of hybridization techniques) is useful for determining
aspects of complexity
o Reannealing the process in which you get two strands of DNA together and form
complimentary gene pairs
5’ A-T-T-C-A-T-G-C-A-T-T-A-G-G-C-T-A-T 3’
3’ T-A-A-G-T-A-C-G-T-A-A-T-C-C-G-A-T-A 5’
Renaturation (reannealing) of denatured DNA depends on many factors, like ionic strength,
length of fragments, temperature, time
These monitor the ability of strands to get back together again
There is a certain procedure used which consists of:
i. Purify DNA, shear to average size of 1000-2000 base pairs
ii. Denature (heat), then allow to reanneal at lower temperature = which allows it to
reanneal
iii. Measure the % reassociated (by absorption) over time
iv. The length of time required for half of the molecules to reanneal is referred to as the
Cot½, and is a measure of complexity
o Cot is the concentration of the fragments and the time required to reanneal; Co
= concentration, t= time
Similar patterns indicative of unique (non-repetitive) sequences
Cot value reflects complexity (genome size and number of each “type” of sequence)
MS-2, T4, and E.coli all have very similar patterns; unique genomes but its taken a bit more
time for E.coli to reanneal because of the genome size
o Genome size of E.coli is 4.3 million base pairs when compared to the others
o MS-2 takes shorter time to renneal since there are more copies
In complex eukaryotic genomes there are three discrete fractions: highly repeated, moderately
repeated, and nonrepeated fraction
A decent analogy for understanding Cot curves is by looking at matching socks: you have one black
sock and need to find a pair:
1. IF drawer contains 500 socks and 400 are black, you’ll have no problem finding a pair quickly
2. IF draw contains 500 socks and 100 are black, takes somewhat longer to find a pair
3. IF drawer contains 500 socks and are black, takes much longer time to find a pair
Equate #socks with # DNA fragments. Some match, some don’t
Only small genome are unique and highly copied
Concentration is important and the time it takes to find a pair (hybridization occurs) is
concentration dependent
Human Genome consists of 22 autosomes and the X/Y sex chromsomes
Haploid genome size is approxiamately 3 x 10^9 base pairs
There are approximately 25,000 genes, of average size of 1.5 base pairs.
Thus about 1.5% of the genome is protein encoding
Genes are interspersed among sequences belonging to families of moderate and highly repetitive
DNA
Graph plots the genome size and the number of
genes; we can assume that humans must be a
lot bigger and the number of genes must be
bigger but mice seem similar
Rice would be assumed to have a small number
of gens but in rice there are about 35,000 genes
compared to humans; rice genome though is tiny
Gets too hot/cold, etc. therefore rice must take
care of itself thus plants do this with these genes
= have the ability to thrive within environment that changes quite often
In situ hybridization is a technique used to discover “gene” location
1. Can get a chromosome from a person which are undergoing mitosis
2. Fix the chromosome by treating the slide with hot salt solution in order for denaturation
3. Two strands move apart from one another and become single stranded DNA
4. Incubate with biotinylated DNA probe, then wash to remove unhybridized DNA. In this example,
satellite DNA is employed.
5. Then you watch as double-stranded DNA hybrid
In order to make a biotinylated probe in which there is a replication in a test tube you must:
1. Assume that you have a cloned DNA sequence
2. You conduct DNA replication and you denature
3. Replication occurs within the test tube and there is a need for AGTC instead you employ a biotin
labeled dCTP which puts in a tag as it goes on = biotinylated probe
4. Incubate with fluorescently labeled avidin to reveal the location of bound, labeled DNA probe
5. Localization of satellite DNA; the counterstain DNA is used to visualize chromosome
6. Localization of satellite DNA can be found in centromeres
Protein in egg white avidly binds to biotin
Fluorescein labeled avidin; it binds tightly to biotin bringing the fluorescent tag
Often, images are superimposed to show relative positions
Alpha satellite DNA found near centromeres
Telomeres are a chromosomes ends