3-Molecular Pathology

Question 1

molecular pathology

definition

Answer 1

discipline within pathology focused on study/diagnosis of disease by examine molecules within organs, cells, body fluids
-subtle changes not detected by cytogenetic

very sensitive so small sample size and work on any cell bc not dep on gene product

Question 2

applications

Answer 2

1. detection of mutations
2. accurate diagnosis and classification of tumors
3. detection of infectious agents
4. tissue typing, paternity testing, forensics

Question 3

mutation types

Answer 3

1. genome = loss/gain of entire chromosome, cytogenetic or molecular analysis
2. chromosome = missing/extra/irregular portion of chromosomal DNA 1+ gene, cytogenetic or molecular
3. gene = complete deletion of 1 gene or single point mutation, molecular only

Question 4

gene mutations

Answer 4

1. point
2. insertions
3. deletions
4. trinucleotide repeat

either in coding or noncoding regions

Question 5

point mutation result

Answer 5

disordered mRNA if @intron

B-thalassemia

Question 6

noncoding region mutations

Answer 6

either dec expression (promoter, enhancer) or unstable mRNA (polyA tail 3’)

B-thalassemia

Question 7

trinucleotide repeat mutation

Answer 7

can be anywhere

fragile X (UTR), huntington disease (exon)

Question 8

direct detection mutation

Answer 8

by comparing with known DNA sequence
3 ways
1. direct sequencing
2. RFLP
3. allele specific extension

Question 9

indirect detection

Answer 9

thru linkage of diseased gene with nearby marker
-site and length polymorphism close to mutation, cuts into pieces
-NOT require gene seq to be known
-requires DNA from family or population to correlate

Question 10

direct sequencing

Answer 10

amplifying region of interest with PCR then sequence the frag
-rapid, accurate, economical

Question 11

RFLP

Answer 11

if mutation alter/destroy a restriction endonuclease site on DNA
-amplify with PCR
-digest PCR product with enzyme
-separate products by electrophoresis gel

Question 12

allele specific extension

Answer 12

fluorescent label nucleotides
-id mutations at specific nucs in early phase of DNA amp by PCR
-inexpensive, automated, fast

detect wild type, mixture, or mutant

Question 13

detecting infectious agents

Answer 13

for COVID:
-use NAAT to detect virus genetic material, more accurate but take longer
OR use antigen test detect viral proteins, not as sensitive

for HIV RNA too

for Hep B and C

Question 14

use PCR for

Answer 14

-gonorrhea
-chlamydial infection
-tuberculosis
-herpes encephalitis

bc very sensitive

Question 15

FISH

Answer 15

detect trisomy 21via interphase nucleus and locus specific probes

Question 16

SKY testing

Answer 16

spectral karyotyping
-visualize all chromosomes by ‘painting’ each pair in diff fluorescent color

detect rearrangments, large deletions/insertions and chromosomal abnormalities bc normal SKY not multicolored
-breast cancer detect

Question 17

array based comparative genomic hybridization

CGH

Answer 17

test genomic DNA and reference DNA
-label with diff red and green dyes then hybridize to slide

yellow = equal amounts of both DNA
green= deletion of DNA from test sample
red = amp of DNA from test smaple
black= no gene detected in both

dark CD44 good, light CD44 bad

Question 18

importance of microarray

Answer 18

predicts survival
-pts with poor outcomes can get early aggressive treatments than waiting for standard therapies to fail

Question 19

forensics and DNA

Answer 19

probes for highly polymorphic regions of genome (MHC)
-PCR and primers amplifies frags from small samples

LADDER = DNA markers for size
K562 = control

can never 100% confirm but can exclude suspects