Biochemistry - Lab Techniques Flashcards
What is the purpose of using DNA polymerase chain reaction?
To amplify the number of copies of DNA fragments
How is the size of a DNA fragment determined from an agarose gel?
A DNA ladder of known lengths is run alongside the polymerase chain reaction fragments
How is DNA separated into two strands during polymerase chain reaction?
By heating
In polymerase chain reaction, premade _____ are used to anneal to specific DNA sequences that will then be amplified.
Primers
What enzyme is used in polymerase chain reaction to amplify DNA?
Heat-stable DNA polymerase
What are the three steps of DNA polymerase chain reaction in order?
Denaturation, annealing, elongation
What is the advantage of using a heat-stable DNA polymerase?
The polymerase is not denatured by heating and can be reused for multiple cycles of polymerase chain reaction
Following PCR, how can products be separated and analyzed?
Agarose gel electrophoresis
Which would travel further on an agarose gel: a 10-kD or an 100-kD DNA fragment?
The 10-kD fragment; smaller molecules travel further
Describe the technique of a Western blot.
Sample protein is separated via gel electrophoresis and transferred to a filter; labeled antibody is used to bind the protein of interest
What blot technique detects specific sequences of DNA?
Southern blot
How does the mnemonic “SNoW DRoP” help one remember which type of blot is used to detect DNA sequences, RNA sequences, or proteins?
Southern = DNA, Northern = RNA, Western = Protein
Describe the technique of a Southern blot.
DNA is run on an electrophoresis gel and transferred to a filter; the DNA on the filter is denatured and exposed to a labeled DNA probe, and then the double-stranded DNA is visualized when the filter is exposed to film
Describe the technique of a Northern blot.
RNA is run on an electrophoresis gel and transferred to a filter; the RNA on the filter is exposed to a labelled DNA probe, and then the hybrid DNA-RNA molecule is visualized when the filter is exposed to film
Which assay would allow you to detect single nucleotide polymorphisms?
Microarrays
How can a microarray profile gene expression levels?
The microarray scanner can detect relative amounts of complementary binding to probes
Describe the technique of using a microarray to detect gene expression levels
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
True or False? In the microarray biology technique, DNA or RNA probes are hybridized to the chip.
True; either DNA or RNA probes may be used
True or False? Enzyme-linked immunosorbent assays test antigen-antibody reactivity.
True
What information would an enzyme-linked immunosorbent assay using a test antibody coupled to a color-generating enzyme give you?
It would tell you whether a certain antigen is present in the patient’s blood
Describe the difference between enzyme-linked immunosorbent assay techniques used to detect antigens and antibodies.
A test antigen labeled with a color-generating enzyme can be used to determine the presence of a specific antibody; a test antibody labeled with a color-generating enzyme can be used to see if a certain antigen is present
Describe the technique of an enzyme-linked immunosorbent assay
A test antigen that is labeled with a color-generating enzyme can be used to determine the presence of a specific antibody; if the antibody is present, the solution will have an intense color reaction
True or False? The sensitivity and specificity of the enzyme-linked immunosorbent assay approaches 100%.
True; however, false-negative and false-positive results may still occur
What is the advantage of a fluorescence in situ hybridization analysis over a karyotype?
It allows researchers to identify anomalies at a molecular level, including deletions that are too small to see on a karyotype
What allows fluorescence in situ hybridization analysis to detect deletions too small to visualize on karyotype?
The fluorescent probes used can bind small segments of DNA and illustrate the presence or absence of genetic material
What laboratory technique is represented with the acronym FISH??
Fluorescent In Situ Hybridization
What does fluorescent in situ hybridization allow researchers to do?
Directly visualize the location of a certain protein or gene on a molecular level through a fluorescent probe that binds to a site of interest
In cloning, what is the significance of inserting DNA to be replicated into plasmids also containing antibiotic-resistance genes and then growing the bacteria on media containing the antibiotic that bacteria is resistant to?
This technique selects for replication of the plasmid containing genes for antibiotic resistance AND the DNA of interest
What is the role of restriction enzymes in DNA cloning?
Restriction enzymes cleave DNA at 4-6 base pair palindromic sequences, allowing for insertion of a fragment into a plasmid
What are the three steps of DNA cloning in order?
Insertion of DNA fragments into bacterial plasmids, cleavage of DNA allowing for insertion, isolation of mRNA to be exposed to reverse transcriptase
What type of genetic material is created by exposing mRNA to reverse transcriptase?
cDNA, which lacks introns
Which type of enzymes cleave DNA at 4- to 6-bp palindromic sequences in cloning?
Restriction enzymes
What distinguishes cDNA from most nuclear DNA?
cDNA lacks introns
Describe the process of Sanger DNA sequencing.
Dideoxynucleotides halt DNA polymerization at each base, generating sequences of various lengths that encompass the entire original sequence so that the terminated fragments are electrophoresed and the original sequence can be deduced
What property of Sanger DNA sequencing is crucial to the ability to determine a DNA sequence using electrophoresis?
A dideoxyribonucleotide must interrupt the sequence at every base in the sequence
Targeted insertion or deletion of a gene into the genome of a mouse occurs through which step?
Homologous recombination
How does an antibiotic-controlled promoter allow you to study genes in which the deletion of that particular gene leads to death of the embryo?
The gene can be manipulated at specific times during development
What is meant by knock-out and knock-in mice?
Knock-out refers to the removal of a gene from a host genome and knock-in refers to the insertion of a gene into a host genome
Describe the process and purpose of RNA inhibition.
The degradation of target mRNA reduces gene expression
Regarding model systems, genes can be manipulated at specific development points using an inducible Cre-lox system with what type of promoter?
Antibiotic-controlled promoter
In RNA inhibition, dsRNA is synthesized then transfected into human cells where it separates and promotes the degradation of which molecule?
mRNA
What phase of mitosis are the chromosomes in when performing a karyotype?
Metaphase
What process compares chromosomes microscopically based on morphology, size, arm-length ratio, and banding pattern?
Karyotyping
What is the diagnostic value of a karyotype?
It can diagnose chromosomal imbalances
Name three types of chromosome imbalances that can be seen on a karyotype.
Autosomal trisomies, microdeletions, and sex chromosome disorders
What tissue can be used to perform a karyotype on a fetus?
You can sample either the amniotic fluid or the placental tissue
What are four tissues that can be used for chromosomal analysis?
Blood, bone marrow, amniotic fluid, and placental tissue
