Lab Techniques

Question 1

What is polymerase chain reaction?

Answer 1

It is a molecular lab procedure used to amplify a desired fragment of dna

Question 2

What are the uses of PCR?

Answer 2

Makes dna from small amount

Determine amount of dna, if it amplifies quickly then large amount of dna was present

Question 3

What are the steps to PCR?

Answer 3

1) denaturation:

DNA is heated at 95 cel to separate the strands

2) annealing

Sample is cooled to 55 cel. The dna primer anneals to the specific sequence to be amplified on each strand

3) elongation

Temp is increased to 72 cel. DNA polymerase attaches the deoxynucleotide triphosphates to the strand to replicate the seq after each primer

Cycle increases until the dna sample size is sufficient

Question 4

What are the clinical correlations of PCR?

Answer 4

HIV viral load:

• Reverse transcriptase makes cDNA
• amplification of cDNA
• amount of dna is directly prop to viral load

Herpes encephalitis

DNA in CSF

Question 5

What does southern blotting recognize?

Answer 5

DNA

Question 6

What aee the steps of southern blotting?

Answer 6

Dna sample is cleaved by restriction enzymes

Separated on a gel by electrophoresis according to size

Transferred to filter paper

Radiolabled cdna prone anneals to complementary strand

Visualisation of dna when exposed to film

Question 7

What are the applications of southern blotting?

Answer 7

Restriction fragment length polymorphisms recognition

For example in sickle cell anemia

Normal B gene: two fragments

1.15, 0.2kb

HBs gene: one fragment

1.35kb

Question 8

What does Northern Blotting detect?

Answer 8

Rna

Question 9

What is application of northern blotting?

Answer 9

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

Question 10

What does western blotting detect?

Answer 10

Protein, labeled antibody binds to revelant protein

Question 11

What is application of western blot?

Answer 11

IgG and IgM lyme disease

IgG in HIV

Question 12

What does southwestern blotting detect?

Answer 12

Dna binding proteins, labelled oligonucleotide probes

Question 13

What is application of southwestern blotting?

Answer 13

Transcription factors

Question 14

What does flow cytometry asses?

Answer 14

Size, granularity, and protein expression of cells

Question 15

What are the cells tagged with in cytometry?

Answer 15

Cells are tagged with flourescent antibody specific to surface protein. Sample is analyzed one cell at a time by focusing a laser on the cell and measuring the light scatter and intensity of fluorescence

Question 16

What is flow cytometry used in?

Answer 16

Paroxysmal nocturnal hemoglobinuria, fetal RBCs in mothers blood, CD4 cell count in HIV

Question 17

What do microarrays consist of?

Answer 17

Thousands of known nucleic acid sequences 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 18

What are microarrays used to asses?

Answer 18

Gene expression levels of thousands of genes simultaneously

Able to detect which SNP (single nucleotide polymorphisms) is present in sample gene and copy number variations (fluorescence intensities of sample and reference is measured and compared. If sample>reference, more copies of genes)

Question 19

What genetic applications can microarray be used in?

Answer 19

Genotyping, clinical genetic testing, forensic analysis, cancer mutations, genetic linkage analysis

Question 20

What is karyotyping?

Answer 20

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

Question 21

What can karyotyping be performed on?

Answer 21

Blood, bone marrow, amniotic fluid, placental tissue

Question 22

What is karyotyping used to diagnose?

Answer 22

Chromosomal abnormalities such as autosomal trisomies, sex chromosome disorders)

Question 23

What is fluorescence in situ hybridisation?

Answer 23

Fluorescent dna or rna probe binds to specific gene site of interest on chromosomes

Question 24

What is FISH used for?

Answer 24

Specific localization of genes and direct visualization of chromosomal anomalies at molecular level

Question 25

What mutations can be found out using FISH?

Answer 25

Microdeletion, no fluorescence on a chromosome compared to fluorescence at the same locus on the second copy of that chromosome

Translocation, fluorescence signal that corresponds to one chromosome is found in a different chromosome

Duplication, a second copy of chromosome, resulting in trisomy or tetrasomy

Question 26

What is ELIZA detecting?

Answer 26

Direct ELIZA: testing for the presence of specific antigen (HBsAg).

• put sample of blood inside the well, the surface antigen sticks to the well surface, and wash away blood. Enzyme linked antibody reacts with the antigen, substrate cleaved gives specfic color

InDirect ELIZA: testing for the presence of antibody (this indirectly testing for the antigen)

-put sample of blood inside the well, the surface antigen sticks to the well surface, and wash away the blood. Antibody not linked to enzyme is added, and the secondary enzyme linked antibody binds to the antibody already bound to antigen

Question 27

Does ELIZA have high specficity and sensitivity?

Answer 27

Yes but less than western bolt (specificity)