Biochemistry Laboratory Techniques

Question 1

Steps to a Polymerase chain reaction (PCR)

Answer 1

1) denature
- DNA sample is heated to 95C to separate DNA strands

2) annealing
- DNA sample is cooled to 55C
- DNA primer, DNA polymerase (Taq) and dNTPs are all added

3) elongation
- temperature is increased to 72C
- DNA polymerase (Taq) attaches dNTPs to the DNA strand to replicate the sequence after each primer

• these steps are contained until amplification of the desired DNA fragment is at sufficient levels*

Question 2

CRISPR/Cas9

Answer 2

A genome editing tool that consists of guide RNA (gRNA) and an endonuclease (Cas9)

Steps:
1) target DNA sequence is added and guide RNA that is artificially constructed matches the target DNA and binds

2) Cas9 makes a brake at the target site which is programmed in the artificial endonuclease

3) is repaired in 1 of two ways:
- break is imperfectly repaired by NHE joining and forms an accidental frame shift mutation (“Knocked-out”)
- donor DNA sequence artificially constructed can be added and fill the gap formed by the break using homology directed repair (HDR) (“knocked-in”)

• note used medically but is promising for future use in deleting negative genes

Question 3

Types of blots

Answer 3

1) southern blot:
- uses DNA to cleave into small pieces, run though gel electrophoresis, and then put into a filter
- filter is then exposed to radiolabled DNA that recognizes and anneals complementary strands
- forms double-DNA strands that have labeled DNA and can be visualized when exposed to film
- * determines DNA disorders and gene properties

2) northern blot
- same as above except for RNA only

3) western blot
- same as above except for proteins and uses antibodies instead of homologous strands of DNA/RNA

4) southwestern blot
- rare use only for DNA-binding protiens

“ SNoW
DRoP”

Question 4

Flow cytometry

Answer 4

Lab technique sued to assess size, granularity and protein expression of cells
-* useful in working up hematologists disorders

Cells are tagged with labeled-antibodies specific to a surface antigen/protein

4 quadrants in the regulating histogram

• bottom left corner = (-) for both proteins measured
• bottom right corner = (+) for X axis protein, (-) for Y axis protein
• upper left corner = (+) for Y axis protein, (-) for X axis protein
• upper right corner = (+) for both proteins measured

Question 5

Enzyme-linked immunosorbant assay (ELISA)

Answer 5

Uses labeled antibodies attached to an enzyme to detect the presence of a specific antigen or antibody in a patients blood sample

• substrate specfic for the enzyme is then added and then the enzyme lights up if positive
• can be direct, indirect or sandwhich forms

*less specific than western blot, but quicker

Question 6

Karyotyping

Answer 6

Measures chromsome structure and constitution by adding colchicine to cultured cells from a blood sample/bone marrow/amnotic fluid or placental tissue
- colchicine stops chromosomes in metaphase and stains them

• used to diagnosis chromosomal abnormalities

Question 7

Fluorescence in situ hybridization

Answer 7

Adding onto karotyping, uses DNA/RNA probes to light up specific gene sites at the molecular level with fluorescent dye.
- used to detect subtle but specific localization fo gene mutations and chromosomal abnormalities

Possible findings:
- microdeletion: no fluorescence on one chromosome compared to a fluorescence seen on the “control” chromosome at the same locus

• translocation: different color fluorescence will appear on a different chromsome than that of the corresponding color (i.e parts of chromsome 17 will show on 19 since 17 parts are different color than 19 indicating a translocation)
• duplication: more than two chromsomes will appear for a specific pair (trisomy or tetrasomy)