Biochem - Lab Techniques

Question 1

What is polymerase chain reaction and its purpose?

Answer 1

Molecular biology laboratory procedure used to amplify a desired fragment of DNA

Question 2

How is polymerase chain reaction used clinically? Give 2 specific examples.

Answer 2

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

Question 3

What are the 3 steps of polymerase chain reaction? Name and briefly describe each.

Answer 3

Steps: (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 DNA polymerase replicates the DNA sequence following each primer; These steps are repeated multiple times for DNA sequence amplification

Question 4

How is agarose gel electrophoresis used? What is used as comparison/reference?

Answer 4

Agarose gel electrophoresis - used for size separation of PCR products (smaller molecules travel further); compared against DNA ladder

Question 5

What are the 4 kinds of blotting procedures?

Answer 5

(1) Southern blot (2) Northern blot (3) Western blot (4) Southwestern blot; Think: “SNoW DRoP: Southern = DNA, Northern = RNA, Western = Protein”

Question 6

Describe the process and result of Southern blot.

Answer 6

A DNA sample is enzymatically cleaved into smaller pieces, electrophoresed on a gel, and then transferred to a filter. The filter is then soaked in a denaturant and subsequently exposed to a radiolabeled DNA probe that recognizes and anneals to its complementary strand. The resulting double-stranded, labeled piece of DNA is visualized when the filter is exposed to film.

Question 7

How does Northern compare/contrast with Southern blot?

Answer 7

Similar to Southern blot, except that an RNA sample is electrophoresed

Question 8

For what metric is Northern blot useful, and why is this important?

Answer 8

Useful for studying mRNA levels, which are reflective of gene expression

Question 9

Describe the process and result of Western blot.

Answer 9

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

Question 10

What is an important clinical correlation for Western blot?

Answer 10

Confirmatory test for HIV after (+) ELISA

Question 11

What does Southwestern blot identify, and how?

Answer 11

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

Question 12

Briefly describe the process and result of microarrays.

Answer 12

Thousands of nucleic acid sequences are arranged in grids on glass or silicon. DNA or RNA probes are hybridized to the chip, and a scanner detects the relative amounts of complementary binding.

Question 13

In general, how are microarrays used clinically?

Answer 13

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

Question 14

What 2 findings are microarrays able to detect? Give 5 applications for such findings.

Answer 14

Able to detect single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) for a variety of applications including (1) genotyping, (2) clinical genetic testing, (3) forensic analysis, (4) cancer mutations, and (5) genetic linkage analysis

Question 15

What is an enzyme-linked immunosorbent assay used to detect?

Answer 15

Used to detect the presence of either a specific antigen (direct) or a specific antibody (indirect) in a patient’s blood sample

Question 16

What distinguishes Indirect from Direct ELISA? Define each as you answer.

Answer 16

Patient’s blood sample is probed with either: (1) Indirect ELISA: uses a test ANTIGEN to see if a specific antibody is present in the patient’s blood; a secondary antibody coupled to a color-generating enzymes is added to detect the first antibody (2) Direct ELISA: uses a test ANTIBODY to see if a specific antigen is present in the patient’s blood; a secondary antibody coupled to a color-generating enzyme is added to detect the antigen

Question 17

What is the result of an enzyme-liked immunosorbent assay if the target substance is present in the sample?

Answer 17

If the target substance is present in the sample, the test solution will have an intense color reaction, indicating a positive test result

Question 18

How is enzyme-linked immunosorbent assay used in laboratories? Give a clinical example.

Answer 18

Used in many laboratories to determine whether a particular antibody (e.g., anti-HIV) is present in a patient’s blood sample.

Question 19

Describe the sensitivity and specificity of ELISA.

Answer 19

Both the sensitivity and specificity of ELISA approach 100%, but both false-positives and false-negative results occur

Question 20

What is the mechanism of Fluorescence in situ hybridization?

Answer 20

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

Question 21

For what purposes is fluorescence in situ hybridization used, and when/why?

Answer 21

Used for specific localization of genes and direct visualization of anomalies (e.g., microdeletions) at molecular level (when deletion is too small to be visualized karyotypes)

Question 22

In Fluorescence in situ hybridization results, what does presence versus absence of fluorescence mean?

Answer 22

Fluorescence = gene is present; No Fluorescence = gene has been deleted

Question 23

Define Cloning.

Answer 23

Clonging is the production of a recombinant DNA molecule that is self perpetuating

Question 24

What are the 5 steps involved in cloning?

Answer 24

Steps: (1) Isolate eukaryotic mRNA (Post-RNA processing steps) of interest (2) Expose mRNA to reverse transcriptase to produce cDNA (lacks introns) (3) Insert cDNA fragments into bacterial plasmids containing antibiotic resistance genes (4) Transform recombinant plasmid into bacteria (5) Surviving bacteria on antibiotic medium produce cDNA

Question 25

In general, what do transgenic strategies in mice involve?

Answer 25

Transgenic strategies in mic involve: (1) Random insertion of gene into mouse genome (2) Targeted insertion or deletion of gene through homologous recombination with mouse gene

Question 26

What is the difference between knock-out and knock-in in gene expression modifications?

Answer 26

Knock-OUT = removing a gene, taking it OUT; Knock-IN = INserting a gene

Question 27

What can Cre-lox system accomplish? Give an example.

Answer 27

Can inducibly manipulate genes at specific developmental points (e.g., to study a gene whose deletion causes embroynic death)

Question 28

What is the process of RNA interference (RNAi)? What does it achieve?

Answer 28

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

Question 29

What is karytoping?

Answer 29

A process in which metaphase chromosomes are stained, ordered, and numbered according to morphology, size, arm-length ratio, and banding pattern

Question 30

What are 4 samples on which karyotyping may be performed?

Answer 30

Can be performed on a sample of blood, bone marrow, amniotic fluid, or placental tissue

Question 31

How is karyotyping used clinically? Give 2 examples.

Answer 31

Used to diagnose chromosomal imbalances (e.g., autosomal trisomies, sex chromosome disorders)