Biochem - Lab Techniques Flashcards
Pg. 81-83 in First Aid 2014 Sections include: -Polymerase chain reaction -Blotting procedures -Microarrays -Enzyme-linked immunosorbent assay -Fluorescence in situ hybridization -Cloning methods -Gene expression modifications -Karyotyping
What is polymerase chain reaction and its purpose?
Molecular biology laboratory procedure used to amplify a desired fragment of DNA
How is polymerase chain reaction used clinically? Give 2 specific examples.
Useful as a diagnostic tool (e.g., neonatal HIV, herpes encephalitis)
What are the 3 steps of polymerase chain reaction? Name and briefly describe each.
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
How is agarose gel electrophoresis used? What is used as comparison/reference?
Agarose gel electrophoresis - used for size separation of PCR products (smaller molecules travel further); compared against DNA ladder
What are the 4 kinds of blotting procedures?
(1) Southern blot (2) Northern blot (3) Western blot (4) Southwestern blot; Think: “SNoW DRoP: Southern = DNA, Northern = RNA, Western = Protein”
Describe the process and result of Southern blot.
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.
How does Northern compare/contrast with Southern blot?
Similar to Southern blot, except that an RNA sample is electrophoresed
For what metric is Northern blot useful, and why is this important?
Useful for studying mRNA levels, which are reflective of gene expression
Describe the process and result of Western blot.
Sample protein is separated via gel electrophoresis and transferred to a filter. Labeled antibody is used to bind to relevant protein.
What is an important clinical correlation for Western blot?
Confirmatory test for HIV after (+) ELISA
What does Southwestern blot identify, and how?
Identifies DNA-binding proteins (e.g., transcription factors) using labeled oligonucleotide probes
Briefly describe the process and result of microarrays.
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.
In general, how are microarrays used clinically?
Used to profile gene expression levels of thousands of genes simultaneously to study certain diseases and treatments
What 2 findings are microarrays able to detect? Give 5 applications for such findings.
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
What is an enzyme-linked immunosorbent assay used to detect?
Used to detect the presence of either a specific antigen (direct) or a specific antibody (indirect) in a patient’s blood sample
What distinguishes Indirect from Direct ELISA? Define each as you answer.
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
What is the result of an enzyme-liked immunosorbent assay if the target substance is present in the sample?
If the target substance is present in the sample, the test solution will have an intense color reaction, indicating a positive test result
How is enzyme-linked immunosorbent assay used in laboratories? Give a clinical example.
Used in many laboratories to determine whether a particular antibody (e.g., anti-HIV) is present in a patient’s blood sample.
Describe the sensitivity and specificity of ELISA.
Both the sensitivity and specificity of ELISA approach 100%, but both false-positives and false-negative results occur
What is the mechanism of Fluorescence in situ hybridization?
Fluorescent DNA or RNA probe binds to specific gene site of interest on chromosomes
For what purposes is fluorescence in situ hybridization used, and when/why?
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)
In Fluorescence in situ hybridization results, what does presence versus absence of fluorescence mean?
Fluorescence = gene is present; No Fluorescence = gene has been deleted
Define Cloning.
Clonging is the production of a recombinant DNA molecule that is self perpetuating
What are the 5 steps involved in cloning?
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
In general, what do transgenic strategies in mice involve?
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
What is the difference between knock-out and knock-in in gene expression modifications?
Knock-OUT = removing a gene, taking it OUT; Knock-IN = INserting a gene
What can Cre-lox system accomplish? Give an example.
Can inducibly manipulate genes at specific developmental points (e.g., to study a gene whose deletion causes embroynic death)
What is the process of RNA interference (RNAi)? What does it achieve?
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
What is karytoping?
A process in which metaphase chromosomes are stained, ordered, and numbered according to morphology, size, arm-length ratio, and banding pattern
What are 4 samples on which karyotyping may be performed?
Can be performed on a sample of blood, bone marrow, amniotic fluid, or placental tissue
How is karyotyping used clinically? Give 2 examples.
Used to diagnose chromosomal imbalances (e.g., autosomal trisomies, sex chromosome disorders)
