Lecture 2 - Southern Blot & Hybirdization

Question 1

what is the purpose of the Southern blot and hybridization technique?

Answer 1

To detect a specific DNA molecule(s) among a complex mixture of DNA molecules

Question 2

Why is it called Southern blot and hybridization?

Answer 2

Because Edward Southern developed the technique.

Question 3

How do you denature double-stranded DNA?

Answer 3

With heat (>95degC) or chemicals (eg. formamide or NaOH)

Question 4

How does renaturation of single-stranded DNA occur?

Answer 4

by cooling or removal of chemicals

Question 5

What can the radioactively-labelled hybridization probe be?

Answer 5

either DNA or RNA

Question 6

What are the characterizations of the Southern blot?

Answer 6

1) Size-fractionation of a complex mixture of DNA molecules by agarose gel-electrophoresis.
2) Denaturation of the double-stranded DNA by NaOH (alkali) to break the hydrogen bond between bases
3) Transfer of the single-stranded DNA molecules to a rigid support, a nitrocellulose filter.

Question 7

What are the steps for filter hybridization?

Answer 7

• high molecular mass DNA to low molecular mass DNA fragments
• single-stranded DNA becomes immoblized to the nitrocellulose filter
• radioactively labelled hybridization probe anneals to target DNA
• wash off probe not hybridized to the target DNA
• overlay filter with X-ray film for autoradiography

Question 8

What creates the photographic emulsion in autoradiogrphy?

Answer 8

• Silver halide (bromide, chloride, iodide) crystals suspended in gelatin on a plastic film

Question 9

What is the chemical process of autoradiogrphy?

Answer 9

Photons or radiation cause silver metallic speck to form. During development, entire crystal is converted into metallic silver that is visible on film

Question 10

What does autoradiography do?

Answer 10

Allows radioactive emissions to activate silver grains in the film. Develops X-ray film

Question 11

In an autoradiogram, all size markers show because they are radioactive; in lanes 2, 3 and so on, only the bands hybridized with what are visible?

Answer 11

hybridized with probe

Question 12

How does the Southern blot and hybridization technique diagnosis sickle-cell anemia?

Answer 12

It detects the Dde I fragments on a Southern blot by probing with beginning of beta-globin gene

Question 13

What is amniocentesis?

Answer 13

A procedure for obtaining fetal cells for genetic testing

Question 14

What DNA probe is used to diagnosis Thalassemias?

Answer 14

Globin genes

Question 15

What DNA probe is used to diagnosis hemophilia?

Answer 15

Factor VIII gene

Question 16

What DNA probe is used to diagnosis dwarfism?

Answer 16

Growth hormone gene

Question 17

What DNA probe is used to diagnosis diabetes?

Answer 17

Insulin gene

Question 18

What DNA probe is used to diagnosis collagen disorders?

Answer 18

collagen gene

Question 19

What DNA probe is used to diagnosis some cancers?

Answer 19

Oncogenes

Question 20

What DNA probe id used to diagnosis Muscular Dystrophy?

Answer 20

MD gene

Question 21

What DNA probe is used to diagnosis Huntington disease?

Answer 21

Huntingtin gene

Question 22

What DNA probe is used to diagnosis cholesterol, metabolism and heart disease?

Answer 22

Low density lipoprotein receptor

Question 23

What DNA probe is used to diagnosis Leukemia and Lymphomas?

Answer 23

Immunoglobulin genes

Question 24

What DNA probe is used to diagnosis emphysema?

Answer 24

Alpha-antitrypsin gene

Question 25

What DNA probe is used to diagnosis immune deficiencies?

Answer 25

T-cell receptor gene

Question 26

What DNA probe is used to diagnosis cystic fibrosis?

Answer 26

CF gene

Question 27

What are the four types of hybridization probes and where do they come from?

Answer 27

1) Messenger RNA that are abundant in particular cells, e.g., globin mRNAs make up 80% of mRNA in red blood cells
2) Heterologous probes, i.e., genes already cloned from related organisms
3) Synthetic DNA probes. Can use reverse genetics to design a DNA probe based on the protein sequence coded by the gene.
4) Anonymous DNA sequences that recognize Restriction Fragment Length Polymorphisms (RFLP)

Question 28

What is the frequency of recombination or crossing over dependent on?

Answer 28

The distance between the gene and the RFLP