Laboratory Techniques in Biochemistry

Question 1

What is PCR?

Answer 1

Polymerase Chain Reaction. Used to amplify a desired segment of DNA.

Question 2

What are the steps of PCR?

Answer 2

1. Denaturation (heat to break apart to two strands)
2. Annealing (during cooling, excess premade DNA primers added and anneal to specific sequences on each strand to be amplified)
3. Elongation (heat stable DNA polymerase replicates DNA sequence following each primer).

Question 3

How are products of PCR separated by size?

Answer 3

Agarose gel electrophoresis. Smaller molecules travel farther.

Question 4

Which blotting technique is associated with material?

Answer 4

SNW DRP: Southern = DNA, Northern = RNA, Western = protein. Southwestern = DNA-binding proteins.

Question 5

What is microarray used for?

Answer 5

Detection of single nucleotide polymorphisms, copy number variants. Profiles gene expression levels of thousands of genes simultaneously to study certain diseases and treatments.

Question 6

What is ELISA?

Answer 6

Used to detect the presence of either a specific antigen or specific antibody in a patient’s blood sample.

Question 7

What is direct ELISA?

Answer 7

Direct ELISA uses a test ANTIBODY to see if specific antigen is present. Antibody directly coupled to color-generating enzyme if antibody binds.

Question 8

What is INdirect ELISA?

Answer 8

Indirect ELISA uses a secondary antibody coupled to a color-generating enzyme - this binds to the antigen/antibody complex.

Question 9

What is karyotyping?

Answer 9

Used to diagnose chromosomal imbalances. Metaphase chromosomes are stained, ordered, numbered according to morphopology, size, arm/length ratio, and banding pattern.

Question 10

What is FISH?

Answer 10

Fluorescence in situ hybridization. Fluorescent DNA/RNA binds to specific gene site of interest on chromosomes. Useful when anomalies are too small to be visualized by karyotyping.

Question 11

What are the steps of cloning?

Answer 11

1. Isolate eukaryotic mRNA.
2. Expose mRNA to reverse transcriptase to produce cDNA (no introns)
3. Insert cDNA fragments into bacterial plasmids containing antibiotic resistance genes.
4. Transform recombinant plasmid into bacteria.
5. Surviving bacteria on antibiotic medium produced cloned DNA.

Question 12

Cre-lox system

Answer 12

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

Question 13

RNA interference

Answer 13

dsRNA produced that is complementary to mRNA of interest. dsRNA separates and promotes degradation of target mRNA, reducing gene expressing.