Lab Techniques Flashcards
What is polymerase chain reaction?
It is a molecular lab procedure used to amplify a desired fragment of dna
What are the uses of PCR?
Makes dna from small amount
Determine amount of dna, if it amplifies quickly then large amount of dna was present
What are the steps to PCR?
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
What are the clinical correlations of PCR?
HIV viral load:
- Reverse transcriptase makes cDNA
- amplification of cDNA
- amount of dna is directly prop to viral load
Herpes encephalitis
DNA in CSF
What does southern blotting recognize?
DNA
What aee the steps of southern blotting?
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
What are the applications of southern blotting?
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
What does Northern Blotting detect?
Rna
What is application of northern blotting?
Useful for studying mrna levels, which are reflective of gene expression
What does western blotting detect?
Protein, labeled antibody binds to revelant protein
What is application of western blot?
IgG and IgM lyme disease
IgG in HIV
What does southwestern blotting detect?
Dna binding proteins, labelled oligonucleotide probes
What is application of southwestern blotting?
Transcription factors
What does flow cytometry asses?
Size, granularity, and protein expression of cells
What are the cells tagged with in cytometry?
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
What is flow cytometry used in?
Paroxysmal nocturnal hemoglobinuria, fetal RBCs in mothers blood, CD4 cell count in HIV
What do microarrays consist of?
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
What are microarrays used to asses?
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)
What genetic applications can microarray be used in?
Genotyping, clinical genetic testing, forensic analysis, cancer mutations, genetic linkage analysis
What is karyotyping?
A process in which metaphase chromosomes are stained, ordered and numbered according to morphology, size, arm-length ratio and banding pattern
What can karyotyping be performed on?
Blood, bone marrow, amniotic fluid, placental tissue
What is karyotyping used to diagnose?
Chromosomal abnormalities such as autosomal trisomies, sex chromosome disorders)
What is fluorescence in situ hybridisation?
Fluorescent dna or rna probe binds to specific gene site of interest on chromosomes
What is FISH used for?
Specific localization of genes and direct visualization of chromosomal anomalies at molecular level
