Biochemistry- Laboratory techniques

Question 1

Polymerase chain

reaction

Answer 1

used to amplify a desired fragment of DNA. Useful as a
diagnostic tool (eg, neonatal HIV, herpes encephalitis).

Question 2

Southern blot

Answer 2

1. DNA sample is enzymatically cleaved, which are separated on a gel by electrophoresis, and then transferred to a filter.
2. Filter is exposed to radiolabeled DNA
   probe that recognizes and anneals to its
   complementary strand.
3. Resulting double-stranded, labeled piece of
   DNA is visualized when filter is exposed to
   film.

Question 3

Northern blot

Answer 3

Similar to Southern blot, except that an RNA
sample is electrophoresed. Useful for studying
mRNA levels

Question 4

Western blot

Answer 4

Sample protein is separated via gel electrophoresis and transferred to a membrane. Labeled antibody is used to bind to relevant protein.

Question 5

Southwestern blot

Answer 5

Identifies DNA-binding proteins (eg, transcription factors) using labeled oligonucleotide probes.

Question 6

SNoW DRoP:

Answer 6

Southern = DNA
Northern = RNA
Western = Protein

Question 7

Flow cytometry

Answer 7

assess size, granularity, and protein expression (immunophenotype) of individual cells in a sample.

Used in workup of hematologic abnormalities and immunodeficiencies.

Data are plotted either as histogram (one
measure) or scatter plot (any two measures).

Question 8

Microarrays

Answer 8

Thousands of nucleic acid sequences are arranged in grids on glass or silicon. Used to profile gene expression levels of thousands of genes simultaneously.

Question 9

Enzyme-linked

immunosorbent assay

Answer 9

used to detect the presence of either a specific antigen (eg, HBsAg) or antibody (eg, anti-HBs) in a patient’s blood sample. less specific than Western blot.

Question 10

Karyotyping

Answer 10

metaphase chromosomes are stained, ordered, and numbered according to morphology, size, arm-length ratio, and banding pattern.

Question 11

Fluorescence in situ

hybridization

Answer 11

Fluorescent DNA or RNA probe binds to
specific gene site of interest on chromosomes

• Microdeletion—no fluorescence on a chromosome.
• Translocation—fluorescence signal that corresponds to one chromosome is found in a different chromosome.
• Duplication—extra site of fluorescence on one chromosome relative to its homologous chromosome.

Question 12

Cloning

Answer 12

Cloning is the production of a recombinant DNA molecule

Question 13

Gene expression

modifications

Answer 13

Knock-out = removing a gene, taking it out.
Knock-in = inserting a gene.