Biochemistry - Laboratory Techniques

Question 1

What is the purpose of polymerase chain reaction?

What are the steps in PCR?

Answer 1

Amplification of desired fragment of DNA.

Useful as a diagnostic tool (e.g. neonatal HIV, herpes, encephalitis)

Steps:

1. Denaturation
2. Annealing- during cooling excess premade DNA primers anneal to a specific sequence on each strand to be amplified
3. Elongation - heat-stable DNA polymerase replicates the DNA sequence following each primer
4. Agarose gel electrophoresis - used for size separation of PCR products and compared against a DNA ladder

Question 2

What is the purpose of a Southern blot?

Answer 2

To visualize double-stranded, labeled pieces of DNA when the filter is exposed to film

Question 3

What is the purpose of a Northern blot?

Answer 3

Basically the same as a Southern blot except an RNA sample is used.

Useful for studying mRNA levels (reflective of gene expression)

Question 4

What is the purpose of a Western blot?

Answer 4

Sample protein separated via gel electrophoresis

Labeled Ab used to bind to relevant protein

Question 5

What is the purpose of a Southwestern blot?

Answer 5

Identifies DNA-binding proteins

Question 6

What is the pneumonic to remember the blots and what they test?

Answer 6

SNoW DRoP

Southern = DNA
Northern = RNA
Western = Protein

Question 7

What is the purpose of a microarray?

Answer 7

Used to profile gene expression levels of thousands of genes simultaneously to study certain diseases

Able to detect SNPs and copy number variations (CNVs)

Question 8

What is the purpose of an enzyme-linked immunosorbent assay?

Answer 8

Used to detect the presence of either a specific Ag (direct) or specific Ab (indirect) in a pt’s blood sample.

Question 9

How does an indirect ELISA work?

Answer 9

Uses a test Ag to see if a specific Ab is present in the patient’s blood.

A second Ab coupled to a color-generating enzyme is added to detect the Ab

Question 10

How does a direct ELISA work?

Answer 10

Uses a test Ab to see if a specific Ag is present in the patient’s blood

A secondary Ab coupled to a color-generating enzyme is added to detect the Ag

Question 11

What is the purpose of fluorescence in situ hybridization (FISH)?

Answer 11

Used for a specific localization of genes and direct visualization of anomalies (e.g. micro deletions) at molecular level (when deletion too small to be seen by karyotype)

Question 12

What is the purpose of cloning?

Answer 12

Production of recombinant DNA molecule that is self perpetuating

Question 13

What is the purpose of RNA interference (RNAi)?

Answer 13

dsRNA synthesized that is complimentary to the mRNA sequence of interest –> transfected into human cells –> darn separates and promotes degradation of target mRNA (“knocking down” gene expression)

Question 14

What is the purpose of karyotyping?

Answer 14

Used to diagnose chromosomal imbalances

Question 15

Codominance

Answer 15

both alleles contribute to the phenotype of the heterozygote

e.g. blood groups

Question 16

Variable expressivity

Answer 16

phenotype varies among individuals w/ same genotype

e.g. 2 pts w/ NF1 have varying disease severity

Question 17

Incomplete penetrance

Answer 17

not all individuals w/ a mutant genotype show the mutant phenotype

e.g. BRCA1 gene mutations do not always result in breast or ovarian cancer

Question 18

Pleiotropy

Answer 18

one gene contributes to multiple phenotype effects

e.g. untreated PKU manifests w/ light skin, intellectual disability, and musty body odor

Question 19

Anticipation

Answer 19

increased severity or earlier onset of disease in succeeding generations

e.g. trinucleotide repeat diseases like Huntington’s Dz

Question 20

Loss of heterozygosity

Answer 20

if pt inherits or develops a mutation in a tumor suppressor gene, the complementary allele must be deleted/mutated before cancer develops (NOT true of oncogenes)

e.g. retinoblastoma and the “two hit hypothesis”

Question 21

dominant negative mutation

Answer 21

exerts a dominant effect

a heterozygote produces a nonfunctional altered protein that also prevents the normal gene product from functioning

Question 22

linkage disequilibrium

Answer 22

tendency for certain alleles at 2 linked loci to occur together more often than expected by chance

Question 23

mosaicism

Answer 23

presence of genetically distinct cell lines in the same individual

arises from mitotic errors after fertilization

e.g. McCune-Albright Syndrome is lethal if the mutation is somatic, but survivable if mosaic

Question 24

locus heterogeneity

Answer 24

mutations at different loci –> similar phenotype

e.g. albinism

Question 25

allelic heterogeneity

Answer 25

different mutations in the same locus –> same phenotype

e.g. beta thalassemia

Question 26

heteroplasmy

Answer 26

presence of both normal and mutated tDNA –> variable expression in mitochondrial inherited disease