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Lecture 48: Cytogenetics & Gene Amplification (part 1), Screen Shot…
Lecture 48: Cytogenetics & Gene Amplification (part 1)
Detection of Cytogenetic Abnormalities
G-Banding Chromosomal Karyotyping
is
Chromosomal Karyotyping
cytogenic technique whereby
number
&
morphology
of nuclear chrom of
any
cell type can be analyzed
Metaphase
chrom grouped by:
-
Size
(largest --> smallest)
-
Centromere position
("p" arm up ; "q" arm down)
-
Exceptions
(chr. 22 > 21; sex chrom last)
Applications
Aneuploidy
(gross/large structural changes)
Diagnosis-
specific
anomalies
Deletions w/
varying sizes
High-Resolution Karyotyping
1,000 band ideogram
Need
dividing
cells!
arrest mitosis in metaphase w/
colchicine
i.e., tumor biopsies, bone marrow, peripheral blood
GTG (G-banding w/trypsin & giemsa banding)
Heterochromatin
stains darkly
gene poor
highly condensed
transcription
silent
Euchromatin
minimal staining
gene rich
less condensed
transc.
active
Standard Karyotype
allows ~450-550 band resolution
around ~150 genes
High-Resolution Chromosomal Banding
More detailed banding! (550-850)
Arrest cell division in
prometaphase & prophase
useful for congenital abnormalities w/finer genetics
FlSH
fluorescence
technique
allows for BOTH
detection + localization
of specific sequence
metaphase FISH
- also says location of genomic region!
Combining:
(1) denatured labeled fluorescent probe &
(2) target DNA -->
direct hybridization w/chrom on slide
Every pair of chrom=
2 dots!
STEPS
STEP 1
DNA probe complementary to gene of interest added to chrom prep
STEP 2
Fluorescent antibody against epitope that was used to tag DNA probe is added & binds to DNA probe
STEP 3
Chrom counterstained w/fluorescent dye of color
different
from that of antibody
--> clear visualization of regions using
fluorescent microscopy!
Applications/Advantages:
Higher
sensitivity, specificity, & resolution vs. banding
Reliable
detection of small microdeletions/microduplications
Can detect aneuploidy + derivative chromosomes
Multiple probes
can be used
DNA/RNA probes
Detects bacteria in clinical samples
Interphase FISH
Fluorescence
in situ
hybridization to human chrom at
metaphase
&
interphase
w/different types of DNA probe
Applications/Advantages:
High
resolution (<1Mb); minimal condensation
Rapid
(<24 hrs;
nondividing
cells;
no
cell culture)
Can use
paraffin-embedded
sections
Valuable-
prenatal diagnosis
Spectral Karyotyping (SKY)
or M-FISH
Applications
complete karyotyping w/automatic analysis
origin of marker chrom
small insertions & complex rearrangements can be inferred
Array-based CGH
Application
patient genome cohybridized w/control reference
query whole genome as ordered array
250 Kb to <25 Kb
However
,
only
relative copy #'s known!
need to be confirmed by karyotyping or FISH
Genome Analysis by
Whole-Genomic Sequencing
Sequencing individual genes to level of nucleotides
Scanning
ENTIRE
genome here
A:
Overrepresetation
of sequence in patient genome -->
duplication
(
aneuploid
)
B:
Patient genome has
contiguous
sequence from both chrom --> shows
Translocation
Detecting Known Mutations
(
+more in part 2)
Other Gene Amplification Techniques
Genomic Library
genomic DNA closed into large insert vectors
BAC's
partial restriction digestion ensures overlapping clones
cDNA Library
collection of clones w/complete genetic repertoire
reverse transcribed from
mRNA
only
contains
spliced exonic regions
tissue-specific expression patterns (
possible
)
expression vectors allow protein product
3 ENZYMES
(1) Reverse Transcriptase
(2) RNAse
(3) DNA polymerase
gene amplication= provide larger quantities of otherwise limited genetic material
Molecular Cloning
Recombinant Genetic Technique
Restriction Endonuclease Digestion
dsDNA cleaving enzyme
recognizes short palindromic sequences
creates restriction fragment mixture w/desired clone --> inserts into compatible cloning vector
Cloning Vector
DNA mol capable of
autonomous
replication in host cell & selected for cloning
ex:
Cloning plasmid
small circular dsDNA mol
replicates independently of host chrom
contains origins of replication, restriction site, selectable markers
Application
Muenke Syndrome
Type of craniosynostosis syndrome defined by single mutation in
FGFR3 gene
that creates new restriction site
DNA clone created from <1 source
clone amplified in living system (ex:
E.coli
)
Manufacturing Human Proteins
large
amounts of human proteins can be made from
protein-coding DNA sequence
inserted into
expression vectors
& introduced into bacterial/yeast/insect/plant/mammalian cells
Examples:
Insulin
Growth factors
Gene Amplification
by
PCR
Polymerase Chain Reaction (PCR)
Application:
PCR to detect Viral Genome in Blood
Blood sample from infected person
Remove cells- centrifugation
Extract RNA
Reverse transcription & PCR amplification of cDNA
Gel electrophorosis
HIV
COVID-19
Amplifies DNA
by
exponentially
increasing DNA amount for analysis (from small clinical sample)
Thermostable DNA polymerase
carefully designed
oligonucleotide
primers
STEPS:
Denaturation, Annealing, Extension
Preparation
isolate genomic DNA
design & synthesize oligo primers
-> flanking DNA sequence of interest
First Cycle
(i) heat denature
dsDNA
(ii) Cool to anneal primers
(iii) extend
Subsequent Cycles
separate, bind, extend (
repeat
)
repeat ~30-35 cycles
--> ~68 billion copies!
5' --> 3' direction
Gel Electrophoresis
Separated
charged samples
of interest based on
size
DNA=
negatively charged!
Smaller= migrates FASTER!
uses
Agarose gel
Negative electrode (top)
Positive electrode (bottom)
Real-time qPCR
RNA isolation & analysis
1st-strand cDNA synthesis
standard PCR amplification
DNA gel electro at
completion
of PCR
Quantification- densitometry
Data analysis
RT-PCR
RNA isolation & analysis
1st strand cDNA synthesis
continuous
fluorescent measurement of
PCR product during
each
cycle
Data analysis
