BIOCHEMISTRY- Laboratory Techniques Flashcards
Which is the fundament for the polymerase chain Reaction?
Molecular biology laboratory procedure used to amplify a desired fragment of DNA
Which are the three steps for the PCR in order to amplify DNA?
Denaturation
Annealing
Elongation
What happens during DNA Denaturarion?
DNA is denatured by heating to generate 2 separate strands
In this phase of PCR, during cooling, excess premade DNA primers anneal to a specific sequence on each strand to be amplified
Annealing
In the Polymerase Chain Reaction, what happens during the elongation phase?
Heat-stable DNA polymerase replicates the DNA sequence following each primer
TRUE OR FALSE… In the PCR the three steps are repeated just once
False, they´re repeated multiple times for DNA sequence amplification
What is the use for aagarose gel electrophoresis?
Used for size separation of PCR products
Which are the Blotting procedures?
Southern Blot
Northern blot
Western Blot
Southwestern Blot
What does Southern blot studies?
DNA
Which is the process followed for DNA studies in Southern blot?
DNA sample is enzymatically cleaved into smaller pieces, electrophoresed on a gel, and then transferred to a filter. In the filter the DNA is denaturant and anneals to its complementary strand. The resulting double stranded is visualized
For what is useful the Northern blot?
RNA is electrophoresed
useful for studying mRNA levels which are reflective of gene expression
What is studied with Western blot?
Proteins
What is used to bind to relevant proteins in Western blot?
Labeled antibody
What importance does a Western blot test has in a certain disease?
Confirmatory test for HIV after + ELISA
So… Southwestern blot, what does it identifies?
DNA-binding proteins using labeled oligonucleotide probes
This laboratory test is able to detect single nucleotide polymorphisms (SNPs) and copy number variations (CNVs)
Microarrays
What does ELISA laboratory test means?
Enzyme linked immunosorbent assay
What is the main purpose of ELISA?
Used to detect the presence of either a specific antigen or a specific antibody in a patient´s blood sample
What does Direct ELISA detects?
antigen
This ELISA test detects the antybody
Indirect ELISA
Direct or Indirect ELISA test… uses a test antigen to see if a specific antibody is present in the patient blood
Indirect ELISA
Direct or Indirect ELISA test… uses a test antibody to see if a specific antigen is present in the patient blood
Direct ELISA
After the first step in ELISA, whats next?
A secondary antibody coupled to a color generating enzyme is added to detect either the antigen or the antibody depending if it is Direct or Indirect ELISA
If the target substance is present in the sample, what are expecting to see?
Intense color reaction
In HIV, what are we looking with ELISA… the antigen or the antibody?
Antibody… Anti-HIV
For ELISA, how is the sensitivity and specifity?
Close to 100%, but both false positive and false negative can occur
When there is a deletion too small to be visualized by karyotype, which test will you use?
Fluorescence in situ hybridization
What is the fundament for the fluorescence in situ hybridization?
Fluorescent DNA or RNA probe bind to specific gene site of interest on chromosomes
For what is useful the flourescence in situ hybriduzation?
Used for specific localization of genes and direct visulization of anomalies Microdeletions
What are the cloning methods?
Cloning is the production of a recombinant DNA molecule that is self perpetuating
Which are the Steps for the cloning method?
- Isolate eukaryotic mRNA of interest
- Expose mRNA to reverse transcriptase to produce cDNA (lacks introns)
- Insert cDNA fragments into bacterial plasmids containing antibiotic resistance genes
- Transform recombinant plasmid into bacteria
- Surviving bacteria on antibiotic medium produce cDNA
What is a Gene expresion modification?
For example, Transgenic strategies in mice involving removing or inserting a gene
In the Gene expression modification, what is a cre-lox modification?
Can inducibly manipulate genes at specific developmental points
In the Gene expression modification, what is RNA interference?
It´s knocking out or removing a gene
Its a process in which methaphase chromosomes are stained, oredered, and numbered acording to morphology, size, arm-lenght ratio, and banding pattern
Karyotyping
From where do we take the samples to do a Karyotyping
Sample of blood, bone marrow, amniotic fluid or placental tissue
What diagnosis use does the karyotype has?
Chromosomal imbalances (autosomal trisomie, sex chromosomes disorders)
What does Southern Blot study?
DNA using DNA probe in Elecrophoresis
What is analized with Northern Blot?
RNA using DNA probe in elecrophoresis
What is analized with Western Blot?
Proteins using 123 I- or enxyme linked antibody
What is analized with Dot (slot) blot?
RNA , DNA or protein
NO ELECROPHORESIS
Its purpose is to determine which restriction fragment of DNA is associated with a particular gene
Southern Blot
Its purpose is to measure size and amounts of specific mRNA molecules to answer questions about gene expression
Northern blot
Its purpose is to measure aoumns of antigen (proteins) or antibodies
Western Blot
Its purpose is to detect specific DNA, RNA, proteins or antibodies
Dot (slot) Blot
What determines which DNA fragments are seen in Southern Blot studies?
Probe
Alternative name for polymorphysms VNTRS (variable number of tandem repeats)
Microsatellite repeats
What does Northern blot measures?
mRNA
mRNA levels
