Biochem Lab Techniques

Question 1

Steps in PCR

Answer 1

1. Denaturation (heating)
2. Annealing (DNA primers anneal to specific sequence on each strand to be amplified)
3. Elongation (heat stable DNA polymerase replicates DNA sequence following each primer)

Question 2

What is PCR useful for?

Answer 2

Used to amplify a specific fragment of DNA; useful as diagnostic tool

Question 3

DNA probe, DNA sample

Answer 3

Southern blot

Question 4

DNA probe, RNA sample

Answer 4

Northern blot

Question 5

What is Northern blot useful for?

Answer 5

Studying mRNA levels (which are reflective of gene expression)

Question 6

Antibody probe, protein sample

Answer 6

Western blot

Question 7

Oligonucleotide probe, DNA binding protein sample

Answer 7

Southwestern blot

Question 8

What are microarrays used for?

Answer 8

Used to profile gene expression levels of thousands of genes simultaneously to study certain diseases and treatments
Able to detects SNPs and copy number variations (CNVs)

Question 9

Used to detect a specific antigen or antibody in a patient’s blood sample

Answer 9

ELISA

Question 10

Difference between direct and indirect ELISA

Answer 10

Direct - use known antibody to identify presence of antigen in patient’s blood; Antibody is directly coupled to color generating enzyme
Indirect - uses test Ag/Ab to detect specific Ag/Ab and then secondary antibody coupled to color generating enzyme is added to detect Ag/Ab complex

Question 11

What is FISH?

Answer 11

Fluorescent DNA/RNA probe binds to specific gene interest on chromosomes –> used for specific localization of genes and direct visualization of anomalies like micro deletions at molecular level

Question 12

Steps of cloning (5)

Answer 12

1. Isolate eukaryotic mRNA of interest
2. Expose mRNA to reverse transcriptase to produce cDNA
3. Insert cDNA fragments into bacterial plasmids containing Ab resistance genes
4. Transform recombinant plasmid into bacteria
5. Surviving bacteria on antibiotic medium produce cloned DNA

Question 13

How is cDNA different than DNA?

Answer 13

It doesn’t contain introns

Question 14

What is cre-lox system?

Answer 14

Can inducibly manipulate genes at specific developmental points to study a gene whose deletion causes embryonic death

Question 15

What is RNA interference?

Answer 15

dsRNA is synthesized that is complementary to target mRNA –> when transfected into human cells the dsRNA separates and promotes degradation of target mRNA –> knocking down gene expression

Question 16

What is codominance?

Answer 16

Both genes contribute to phenotype of heterozygote (blood groups, alpha 1 antitrypsin deficiency)

Question 17

What is variable expressivity?

Answer 17

Phenotype varies among individuals with same genotype (2 patients with NF1 have varying severity)

Question 18

What is incomplete penetrance?

Answer 18

Not all individuals with mutant genotype show the mutant phenotype (BRCA1 mutation)

Question 19

What is Pleiotropy?

Answer 19

One gene contributes to multiple phenotypic effects (PKU causes light skin, intellectual disability, musty body odor)

Question 20

What is Anticipation?

Answer 20

Increased severity of earlier onset in succeeding generations (Trinucleotide repeat diseases)

Question 21

What is loss of heterozygosity?

Answer 21

It a patient inherits/develops a mutation in a tumor suppressor gene, the complementary allele must be deleted or mutated before cancer develops (two hit hypothesis)

Question 22

Two hit hypothesis is an example of?

Answer 22

Loss of heterozygosity

Question 23

What is dominant negative mutation?

Answer 23

Exerts a dominant effect; a heterozygote produces a non-functional altered protein that also prevents the normal gene product from functioning (mutation of transcription factor in its allosteric site that prevents wild type transcription factor from binding)

Question 24

What is linkage disequilibrium?

Answer 24

Tendency for certain alleles at 2 linked loci to occur together more or less often than expected by chance (measured in population)

Question 25

What is mosaicism?

Answer 25

Presence of genetically distinct cell lines in same individual

1. Somatic mosaicism = mutation arises from mitotic errors after fertilization and propagates through multiple tissues/organs
2. Gonadal mosaicism = mutation only in egg/sperm cells = mutation only in offspring

Question 26

Example of mosaicism?

Answer 26

McCune Albright syndrome (lethal if mutation occurs before fertilization)

Question 27

What is locus heterogeneity?

Answer 27

Mutations at different loci can produce a similar phenotype - Albinism

Question 28

What is allelic heterogeneity?

Answer 28

Different mutations in same locus produce the same phenotype - B thalassemia

Question 29

What is heteroplasmy?

Answer 29

Presence of both normal and mutated mtDNA; resulting in variable expression in mitochondrially inherited disease`

Question 30

What is uniparental disomy?

Answer 30

Offspring receives 2 copies of chromosome from one parent and no copies from the other

Question 31

What is uniparental heterodisomy?

Answer 31

Meiosis I error - offspring receives 2 copies of chromosome from one parent (same chromosomes as parent)

Question 32

What is uniparental isodisomy?

Answer 32

Meiosis II error or post zygotic chromosomal duplication of one pair of chromosomes (2 copies are identical!)

Question 33

Hyperphagia, obesity, intellectual disability, hypogonadism and hypotonia

Answer 33

Prader Willi syndrome (maternal imprinting; paternal gene is mutated/deleted OR maternal uniparental disomy)

Question 34

Inappropriate laughter, seizures, ataxia, severe intellectual disability

Answer 34

Angelman syndrome (paternal imprinting, maternal gene is deleted/mutated)

Question 35

Inheritance pattern of enzyme deficiencies (usually)

Answer 35

Autosomal recessive

Question 36

Inheritance pattern of defects in structural genes

Answer 36

Autosomal dominant

Question 37

Inheritance pattern of hypophosphatemic rickets

Answer 37

X linked dominant

Question 38

Conditions that are polygenic

Answer 38

Androgenetic alopecia
Epilepsy
Ischemic heart disease
Schizophrenia
Type II DM
HTN
Glaucoma
Malignancy

Question 39

Mutation causing ADPKD

Answer 39

Autosomal dominant
85% due to mutation in PKD1 on chromosome 16
Remainder from PKD2 on chromosome 4

Question 40

Mutation causing Familial adenomatous polyposis

Answer 40

Autosomal dominant

Mutation in chromosome 5q (APC gene)

Question 41

Mutation in Familial hypercholesterolemia

Answer 41

Autosomal dominant

Defective or absent LDL receptor –> elevated LDL

Question 42

Mutation in Hereditary hemorrhagic telangiectasia

Answer 42

Also known as Osler-Weber-Rendu syndrome

Autosomal dominant

Question 43

Mutation in Hereditary Spherocytosis

Answer 43

Autosomal dominant

Mutation in spectrin or ankyrin

Question 44

Mutation in Huntington disease

Answer 44

Autosomal Dominant

on Chr 4; trinucleotide repeat CAG

Question 45

Mutation in Li Fraumeni

Answer 45

Autosomal Dominant
Abnormal Tp53 (sarcoma, breast, leukemia, adrenal gland cancer)

Question 46

Mutation in Marfan’s

Answer 46

Autosomal dominant

FBN1 mutation on chromosome 15 –> defective fibrillin

Question 47

Mutation causing MEN2A and MEN2B

Answer 47

Autosomal dominant

Mutation in RET gene

Question 48

Mutation causing NF1 (von Recklinghausen disease)

Answer 48

Autosomal dominant

Mutation in NF1 gene on chromosome 17

Question 49

Mutation causing NF2

Answer 49

Autosomal dominant

Mutation in NF2 gene on chr. 22

Question 50

Mutation causing tuberous sclerosis

Answer 50

Autosomal dominant

Question 51

Mutation causing von Hippel Lindau disease

Answer 51

Autosomal dominant

deletion of VHL gene on chromosome 3p