BIOCHEM--Lab Techniques

Question 1

Polymerase Chain Reaction: what is it?

Answer 1

procedure to amplify desired fragment of DNA

Useful as diagnostic–neonatal HIV, herpes encephalitis

Question 2

Polymerase Chain Reaction: Steps

Answer 2

1. denaturation
2. annealing
3. elongation

Question 3

Polymerase Chain Reaction: denaturation

Answer 3

DNA heated to ~95C to separate the strands

Question 4

Polymerase Chain Reaction: annealing

Answer 4

sample cooled to ~55C
add DNA primers, a heat stable DNA polymerase (Taq), and dNTPs
Primers anneal to specific sequence to be amplified

Question 5

Polymerase Chain Reaction: elongation

Answer 5

temperature increased to ~72C

DNA polymerase attaches dNTPs to the strand to replicate the sequence that follows the primer

Question 6

CRISPR/Cas9: what is it?

Answer 6

a genome editing tool derived from bacteria

Question 7

CRISPR/Cas9: what is it comprised of and what do these components do?

Answer 7

1. an endonuclease (Cas9): cleaves the dsDNA

2. a guide RNA (gRNA): binds complementary target DNA sequence. can be designed to target any DNA sequence

Question 8

CRISPR/Cas9: after the system introduces a gap in the DNA, how is it filled in?

Answer 8

1. by cell DNA repair machinery (nonhomologous end joining): called “Knock out”
   or
2. by donor DNA that can be added to the system: called “knock in”

Question 9

Blotting procedures: “SNoW DRoP” pneumonic

Answer 9

Southern = DNA
Northern = RNA
Western = Protein

Question 10

How to do a Southern Blot

Answer 10

1. DNA sample enzymatically cleaved into small pieces that are then separated on gel by electrophoresis, then transferred to a filter.
2. filter exposed to radio-labeled DNA probe that recognizes and binds to complementary strand
3. resulting double-stranded, labeled piece of DNA is visualized when filter exposed to film.

Question 11

How to do a Northern Blot

Answer 11

1. same as southern blot, but use RNA instead

Question 12

What is Northern Blot useful for?

Answer 12

studying mRNA levels, which are reflective of gene expression

Question 13

How to do a Western Blot

Answer 13

1. protein sample separated via gel electrophoresis and transfered to a membrane
2. labeled antibody is used to bind relevant protein

Question 14

WTF is a southwestern blot?

Answer 14

a test used to identify DNA-binding proteins like transcription factors by using labeled oligonucleotide probes.

Question 15

Flow Cytometry: what is it?

Answer 15

test to assess the size, granularity, and protein expression of individual cells in a sample.

Question 16

Flow Cytometry: commonly used in workups for ___ abnormalities and ___

Answer 16

hematologic and immunodeficiencies

Question 17

Flow Cytometry: how does it work?

Answer 17

1. cells are tagged with ab specific to surface or intracellular proteins.
2. those ab are then tagged with unique fluorescent dye.
3. sample analyzed one cell at a time w/ a laser that measures light scatter and intensity of the fluorescence

Question 18

Flow Cytometry: how is data from this analysis plotted?

Answer 18

as a histogram (one measure)
or
scatter plot (any two measures)

Question 19

What are microarrays used for?

Answer 19

used to profile gene expression levels of thousands of genes at the same time to study certain diseases and treatments.