Week 11 Flashcards
Chromosomal Rearrangements, Transposable Elements, and DNA Analysis
define: chromosomal rearrangements
mutations occurring on a larger scale
is homozygosity for gene deletions always lethal
no, but it usually is
is heterozygosity for gene deletions lethal
it is usually detrimental
define: gene dosage
the number of times a given gene is present in the genome
what causes cri-du-chat syndrome
deletion on the short arm of chromosome 5
define: tandem duplications
duplications that lie adjacent to each other
can a tandem duplication be in reverse
yes
define: nontandem duplications
two or more copies of regions that are not adjacent to each other
do nontandem duplications occur on the same chromosome
they can be on the same chromosome or different chromosomes
do duplications have obvious phenotypic consequences
many do not
what does change in phenotype depend on for duplications
whether there is a gene-dosage effect for that genes product
what causes Fragile X syndrome
large number of duplications of a CGG repeat on the X chromosome
why is the CGG on the X chromosome for fragile X syndrome harmful
prevents the gene downstream of that region from being transcribed
what does unequal crossing between duplications cause
increases of decreases in gene copy number
how was myoglobin formed
duplication of β -globin gene and subsequent selection
what is the advantage of myoglobin
has a greater affinity for O2 than hemoglobin
do inversions and translocations result in a change of genetic content
no, it is just a change of distribution
when do inversions occur
- following two double strand breaks
- from rare crossovers between related DNA sequences present in two different locations on the same chromosome
define: paracentric inversions
exclude the centromere
define: pericentric inversions
include the centromere
how do chromosome 4 in humans and chimpanzees compare
differ by only a pericentric inversion
when do mutant phenotypes occur with inversions
if one end lies within a coding region of DNA
define: translocations
large scale chromosomal rearrangements in which part of one chromosome either becomes attached to another chromosome or parts of two different chromosomes trade places
define: reciprocal translocation
parts of two different chromosomes trade places
define: robertsonian translocation
reciprocal translocations arising from breaks at or near the centromeres of two acrocentric chromosomes
what is the result of robertsonian translocation
one large and one small chromosome, small chromosome is usually lost
what causes Familial Down’s syndrome
robertsonian translocation between C14 and C21 in the germ cell mated with a normal gamete
why is there a difference in chromosome number between humans and the great apes
robertsonian translocation occurred sometime in our evolutionary history
define: transposable elements
small segments of DNA which are able to move from one position to another in the genome
how much of the human genome consists of transposable elements
40%
who inferred the presence of transposable elements
Rhoades and McClintock
what does it mean for a transposable element to be selfish
only carries information required for their own propagation
what are the two kinds of transposable elements in prokaryotes
- insertion sequences (IS)
- transposons (Tn)
how do insertion sequences move
by cut and paste
what do insertion sequences contain
terminal inverted repeats
what do insertion sequences code for
a transposase enzyme
what does transposase do
recognizes inverted repeats and cuts the host DNA for IS movement
what are the steps of insertion of IS into target DNA
1) staggered cleavage of target DNA
2) insertion of IS
3) synthesis of DNA fills gap
how do transposons move
by cut and paste
what do transposons code for
a transposase enzyme and other genes such as resistance genes
define: composite Tns
2 IS elements flanking one or two genes
what are the two kinds of transposable elements in eukaryotes
- transposons
- retrotransposons
how do retrotransposons move
makes a copy of itself
how do retrotransposons move
via an RNA intermediate
how is the RNA intermediate of a retrotransposon inserted into the DNA
reverse transcriptase copies the RNA into a double-stranded DNA molecule for insertion into the genome
what are the two major classes of retrotransposons in mammals
- LINEs (long interspersed elements)
- SINEs (short interspersed elements)
what is often the cause of unequal crossovers
transposable elements
what is an example of a result of transposable element insertions
yellow cauliflower
what are DNA probes
short single-stranded stretches of DNA of known composition
how long are DNA probes
from 25 to several thousand nucleotides
how are DNA probes visualized
they are labelled with 32P or fluorescent dyes
what are DNA probes used for
identification of clones that contain complementary DNA sequences
what are the steps of screening libraries by hybridization
1) plate bacteria carrying recombinant DNA and incubate until colonies form
2) replica-plate colonies
3) lyse cells, denature and bind DNA to the membrane
4) add DNA probe and incubate under annealing conditions
5) rinse membrane to remove non-annealing DNA
6) expose to X-ray film
7) pick corresponding colony from plate and isolate recombinant DNA
what do dark spots on X-ray show
recombinant, positive hybridization
what are the requirements of hybridization probes
- region of complementarity between probe and target sequence must be long enough to allow sufficient hydrogen bonds to provide a cohesive force
In General - longer than 50-100 bp
- complementarity is more than 80%
what is southern blot analysis used for
showing the location of a gene within a larger fragment of DNA
what are the steps of southern blot analysis
1) transfer DNA fragments from agarose gel to nitrocellulose filter paper
2) perform hybridization between a labelled probe and DNA on the filter
3) expose to X-ray film to identify hybrid
what is a use of southern blot analysis
identification of patients with tuberculosis
who invented polymerase chain reaction (PCR)
Dr. Kary Mullis
what is the method of PCR
1) design two primers that are complementary to each end of the sequence of interest
2) mix the primers, template DNA, thermostable DNA polymerase, and the 4 nucleotide bases in a microtube
3) begin thermocycling procedure
what are the steps of thermocycling for PCR
1) denature: 94°C for 5 min
2) anneal: 50-60°C for 2 min
3) extend: 72°C for 2-5 min
(repeat)
how does PCR work so quickly
each cycle of PCR results in an exponential duplication of the strands
where is PCR used
- diagnosis of infection
- paleomolecular biology
- forensic science
what is the basis of sanger dideoxy sequencing
based on lack of 3’ OH group to terminate chain elongation
how does sanger dideoxy sequencing work
- mix DNA polymerase with dATP, dGTP, dCTP, dTTP
- put in tubes with ddA, ddG, ddC, and ddT that are uniquely labelled
- separate strands on polyacrylamide gel
why is polyacrylamide gel used in sanger dideoxy sequencing
it can resolve strands that differ in length by a single nucleotide
how is DNA sequencing done in modern times (sanger dideoxy sequencing)
uses a laser and fluorescence detector on the gel
how is genetic testing for sickle-cell done
- PCR amplification of 500 bp fragment of interest then REN digestion with MstII
- normal allele will be cut and give 2 bands, sickle cell allele will only give one band
how is preimplantation genetic testing for cystic fibrosis done
- retrieve several ripe eggs from ovary, fertilize with sperm
- at 6-10 cell stage, remove one cell from each embryo for genetic testing
- lyse cell, isolate DNA
- perform PCR amplification of site of common mutation
- hybridize half with normal probe and other half with mutant probe
- dark spot of X-ray means positive hybridization
what are the canadian genetic testing regulations
allows licensing of tests for genetic diseases based on:
1) high penetrance (90% threshold)
2) cannot be successfully treated
3) are of early onset
