Lab Techniques

Question 1

What is the Polymerase Chain Reaction technique used for?

Answer 1

To amplify a desired fragment of DNA (p.79)

Question 2

Name the three steps of PCR.

Answer 2

1.) Denaturation; 2.) Annealing; 3.) Elongation. These steps are repeated many times (p.79)

Question 3

Describe the first step of PCR.

Answer 3

Denaturation- DNA is denatured by heating to generate 2 separate strands (p.79)

Question 4

Describe the second step of PCR.

Answer 4

Annealing- during colling, excess premade DNA primers anneal to a specific sequence on each strand to be amplified (p.79)

Question 5

Describe the third step of PCR.

Answer 5

Elongation- heat stable DNA polymerase replicates the DNA sequence following each primer (p.79)

Question 6

How are PCR products separated by size?

Answer 6

Agarose gel electrophoresis. Smaller molecules travel further and their size is compared against a DNA ladder (p.79)

Question 7

Describe the process of a southern blot.

Answer 7

A DNA sample is electrophoresed on a gel and transferred to a filter. The filter is soaked in denaturant and 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 (p.80)

Question 8

What does a Southern blot detect? A Western? A Northern?

Answer 8

Southern- DNA; Northern- RNA; Western- Protein (p.80)

Question 9

Describe the process of a northern blot.

Answer 9

Similar to southern blot but an RNA sample is electrophoresed (p.80)

Question 10

What is a northern blot useful for studying?

Answer 10

mRNA levels (p.80)

Question 11

Describe the process of a western blot.

Answer 11

Sample protein is separated via gel electrophoresis and transfererd to a filter. Labeled antibody is used to bind relevant protein (p.80)

Question 12

What does a southwestern blot identify?

Answer 12

DNA binding proteins (e.g. transcription factors) using labelled oligonucleotide probes (p.80)

Question 13

Describe the process of a microarray.

Answer 13

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 relative amounts of complementary binding (p.80)

Question 14

What are microarrays used to measure?

Answer 14

Used to profile gene expression levels of thousands of genes simultaneously to study diseases and treatments (p.80)

Question 15

What are microarrays capable of detecting?

Answer 15

Single nucleotide polymorphisms (SNPs) for a variety of applications including genotyping, forensic analysis, predisposition to disease, cancer mutations, and genetic linkage analysis (p.80)

Question 16

What is an ELISA?

Answer 16

A rapid immunologic technique used for detecting antigen-antibody activity in a patient’s blood. If a target substance is present in a sample, the test solution will have a positive colour reaction (p.80)

Question 17

What are ELISAs commonly used for?

Answer 17

To determine if a particular antibody is present in a patient’s blood (anti-HIV, etc) (p.80)

Question 18

Are ELISAs a sensitive and specific test?

Answer 18

Yes, sensitivity and specificity approach 100% but false positive and false negatives do occur (p.80)

Question 19

What is an indirect ELISA?

Answer 19

Uses a test antigen to see if a specific antibody is present in a patient’s blood. A secondary antibody is coupled to a colour generating enzyme is added to detect the first antibody (p.80)

Question 20

What is a direct ELISA?

Answer 20

Uses a test antibody coupled to a color-generating enzyme to see if a specific antigen is present in the patient’s blood (p.80)

Question 21

Describe the process of Flourescence in situ Hybridization (FISH).

Answer 21

Flourescent DNA or RNA probe binds to specific gene site of interest on chromosomes. Flourescence indicates that a gene is present, no flourescence indicates that a gene has been deleted (p.81)

Question 22

What is the FISH procedure used for?

Answer 22

To localize specific genes and to directly visualize anomalies (e.g. microdeletions) at a molecular level when deletion is too small to be visualized by karyotype (p.81)

Question 23

What is DNA cloning?

Answer 23

The proudction of a recombinant DNA molecule that is self perpetuating (p.81)

Question 24

Describe the four steps of cloning.

Answer 24

1.) Isolate eukaryotic mRNA (post mRNA processing steps) of interest; 2.) expose the 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 (p.81)

Question 25

Describe two ways that mice can be transgenically altered.

Answer 25

1.) random insertion of gene into mouse genome; 2.) target insertion or deletion of gene through homologous recombination with mouse gene (p.81)

Question 26

What is the Cre-lox system of gene expression modification?

Answer 26

Can manipulate genes at specific developmental points using an antibiotic promoter (e.g. to study a gene whose deletion causes embryonic death) (p.81)

Question 27

What is RNA interference (RNAi)?

Answer 27

dsRNA is synthesized that is complementary to the mRNA sequence of interest. This is transfected into human cells and dsRNA separates and promotes degradation of target mRNA, knocking down gene expression (p.81)

Question 28

Describe the process of karyotyping?

Answer 28

A process in which metaphase chromosomes are stained, ordered, and numbered according to morphology, size, arm-length ratio, and banding pattern. It can be performed on a sample of blood, bone marrow, amniotic fluid, or placental tissue (p.81)

Question 29

What is karyotyping used for?

Answer 29

Used to diagnose chromosomal imbalances (e.g. autosomal trisomies, sex chromosome disorder) (p.81)