Fa: Biochemistry - Lab Techniques

Front 1

What is the function of the Polymerase Chain Reaction?

Back 1

Molecular biology laboratory procedure used to amplify a desired fragment of DNA

Front 2

What are the steps of the Polymerase Chain Reaction?

Back 2

1. Denaturation
2. Annealing
3. Elongation

Steps are repeated multiple times for DNA sequence amplification

Front 3

What happens in the first step of the Polymerase Chain Reaction?

Back 3

Denaturation
- DNA is denatured by heating
- Generates 2 separate strands

Front 4

What happens in the second step of the Polymerase Chain Reaction, after Denaturation?

Back 4

Annealing
- During cooling, excess pre-made DNA primers anneal to specific sequence on each strand to be amplified

Front 5

What happens in the third step of the Polymerase Chain Reaction, after Annealing?

Back 5

Elongation
- Heat-stable DNA polymerase replicates the DNA sequence following each primer

Front 6

What can you do after amplifying your DNA product via the Polymerase Chain Reaction?

Back 6

Agarose Gel Electrophoresis:
- Size separation of PCR products (smaller molecules travel further)
- Compare against a DNA ladder

Front 7

What are the types of Blotting procedures?

Back 7

- Southern Blot
- Northern Blot
- Western Blot
- Southwestern Blot

Front 8

How do you remember what the different Blotting techniques sample?

Back 8

SNoW DRoP
- Southern: DNA
- Northern: RNA
- Western: Protein

(Southwestern: DNA-binding proteins)

Front 9

What does a Southern Blot sample? Procedure?

Back 9

DNA sample:
- Electrophorese sample on a gel and transfer to a filter
- Filter is soaked in denaturant and exposed to a radiolabeled DNA probe
- DNA probe recognizes and anneals to its complementary strand
- Resulting labeled piece of dsDNA is visualized when the filter is exposed to film

Front 10

What does a Northern Blot sample? Procedure?

Back 10

RNA sample:
- Similar to Southern blot procedure
- Sample is electrophoresed
- Useful for studying mRNA levels

Front 11

What does a Western Blot sample? Procedure?

Back 11

Protein sample:
- Sample is separated via gel electrophoresis and transfered to a filter
- Labeled antibody is used to bind to relevant protein

Front 12

What does a Southwestern Blot sample? Procedure?

Back 12

Identifies DNA-Binding Proteins (eg, transcription factors):
- Use labeled oligonucleotide probes

Front 13

What does a Microarray sample? Procedure?

Back 13

Nucleic Acid Sequences:
- Thousands of nucleic acid sequences are arranged in grids on glass or silicon
- DNA or RNA probes are hybridized to the chip
- Scanner detects the relative amounts of complementary binding

Front 14

What is the function of a Microarray?

Back 14

- Profile gene expression levels of thousands of genes simultaneously to study certain diseases and treatments
- Able to detect single nucleotide polymorphisms (SNPs) for a variety of applications, including genotyping, forensic analysis, predisposition to disease, cancer mutations, and genetic analysis

Front 15

Which lab technique can detect single nucleotide polymorphisms (SNPs)? What purpose?

Back 15

Microarray
- Genotyping
- Forensic analysis
- Predisposition to disease
- Cancer mutations
- Genetic analysis

Front 16

What does an Enzyme-Linked Immunosorbent Assay (ELISA) sample? Procedure?

Back 16

Antigen-Antibody Reactivity:
- Rapid immunologic technique
- Blood sample is probed either with indirect ELISA (test antigen) or direct ELISA (test antibody)
- If the target substance is present in the sample, test solution will have an intense color reaction, indicating a positive test

Front 17

What does ELISA stand for? What are the two types? How do they differ?

Back 17

Enzyme-Linked Immunosorbent Assay:
- Indirect: use a test antigen to see if a specific antibody is present in the patient's blood, a secondary antibody coupled to a color generating enzyme is added to detect the first antibody

- Direct: use a test antibody coupled to a color-generating enzyme to see if a specific antigen is present in the patient's blood

Front 18

What is the purpose of Enzyme-Linked Immunosorbent Assay (ELISA)?

Back 18

- Determine whether a particular antibody (eg, anti-HIV) is present in a patient's blood sample
- Both sensitivity and specificity of ELISA approach 100% but both false-positive and false-negative results do occur

Front 19

What does an Fluorescence In Situ Hybridization (FISH) sample? Procedure?

Back 19

Looking for specific gene site of interest on chromosomes:
- Fluorescent DNA or RNA probes bind to these specific genes
- Used for specific localization of genes and direct visualization of anomalies (eg, microdeletions) at molecular level (when deletion is too small to be visualized by karyotype)
- Fluorescence = gene is present
- No fluorescence = gene has been deleted

Front 20

What does FISH stand for? Purpose?

Back 20

Fluorescence In Situ Hybridization:
- Used for specific localization of genes and direct visualization of anomalies (eg, microdeletions) at molecular level (when deletion is too small to be visualized by karyotype)

Front 21

What does cloning do?

Back 21

Produces a recombinant DNA molecule that is self-perpetuating

Front 22

What are the steps for cloning?

Back 22

1. Isolate eukaryotic mRNA (post-RNA processing steps) of interest
2. Expose mRNA to reverse transcriptase to produce cDNA
3. Insert cDNA fragments into bacterial plasmids containing antibiotic resistance genes
4. Surviving bacteria on antibiotic medium produce cDNA library

Front 23

What are the gene expression modification strategies used in mice?

Back 23

Transgenic strategies in mice involve:
- Random insertion of gene into mouse genome
- Targeted insertion or deletion of gene through homologous recombination with mouse gene

Front 24

What can the Cre-Lox system do? Example?

Back 24

- Inducibly manipulate genes at specific developmental points using an antibiotic-controlled promoter
- Eg, to study a gene whose deletion causes embryonic death

Front 25

What can the RNA interference (RNAi) system do?

Back 25

- 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
- This knocks down gene expression

Front 26

What is the sample for karyotyping? Procedure?

Back 26

Performed on a sample of blood, bone marrow, amniotic fluid, or placental tissue

Metaphase chromosomes are:
- Stained
- Ordered
- Numbered according to morphology, size, arm-length ratio, and banding pattern

Front 27

What is the purpose of karyotyping?

Back 27

Diagnose chromosomal imbalances (eg, autosomal trisomies, sex chromosome disorders)