Biochemistry-Laboratory Technique

Question 1

Name and explain the three steps of PCR.

Answer 1

1. Denaturation- DNA is denatured by heating to generate 2 separate strands.
2. Annealing- during cooling, excess premade DNA primers anneal to a specific sequence on each strand to be amplified.
3. Elongation- heat-stable DNApolymerase replicates the DNA sequence following each primer.

Question 2

True or False: Agarose gel separates PCR products based on size.

Answer 2

True. Larger molecules move slower down the agarose whereas smaller molecules move further.

Question 3

What is the function of a PCR?

Answer 3

Amplify a small specific segment of DNA. It is a useful tool for diagnosing neonatal HIV and herpes encephalitis

Question 4

What is the function of a Southern blot?

Answer 4

Identifying DNA by exposing it to a filter with probes

Question 5

What molecule does a Northern blot identify?

Answer 5

RNA

Question 6

What molecule does a Western blot identify?

Answer 6

Protein

Question 7

What molecule does a Southwestern blot identify?

Answer 7

DNA-binding proteins

Question 8

What diagnostic tool can profile gene expression levels of thousands of genes simultaneously to study certain diseases and treatments. Hint: it is also able to detect single nucleotide polymorphisms and copy number variations.

Answer 8

Microarrays

Question 9

What diagnostic tool is used to detect the presence of either a specific antigen (directly) or a specific antibody (indirectly) in a patient’s blood sample?

Answer 9

Enzyme-Linked ImmunoSorbent Assay (ELISA). Used in laboratories to determine whether a particular antibody is present specificity is close to 100%

Question 10

What is the function of fluorescence in situ hybridization?

Answer 10

Fluorescent DNA or RNA probe binds to specific gene site of interest on chromosome. Used for specific localization of genes and direct visualization of anomalies.

Question 11

If there is no fluorescence in a fluorescence in situ hybridization, was there a duplication?

Answer 11

No most likely the gene was deleted

Question 12

What are the steps of cloning?

Answer 12

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

Question 13

What do we mean when we say knock-out or knock-in?

Answer 13

It is a form of gene expression modification that results in removal of a gene or insertion of a gene, respectively.

Question 14

What is the function of Cre-lox system?

Answer 14

TO study and inducibly manipulate genes at specific developmental points(e.g. to study a gene whose deletion causes embryonic death)

Question 15

What is RNA interference used for?

Answer 15

dsRNA is synthesized that is complementary to the mRNA sequence of interest. When transferred into human cells, dsRNA separates and promotes degradation of target mRNA, “knocking down” gene expression

Question 16

What is often used to diagnose chromosomal imbalances?

Answer 16

Karyotyping