Wright Lecture 2: Southern Blot and Hybridization

Question 1

What is the purpose of the Southern Blot and Hybridization technique?

Answer 1

To detect a specific DNA molecule among a complex mixture of DNA molecules
Find specific sequences in thousands of fragments of DNA

Question 2

Edwin Southern

Answer 2

Developed Southern blot, published 1975

Question 3

Hybridization

Answer 3

1. Denaturation of double stranded DNA

2. Detection of specific DNA molecule in complex mixture of DNA molecules by using hybridization probe

Question 4

Liquid hybridization

Answer 4

1. Denaturing of dsDNA

Adding reactively labelled probe that binds to sequence in DNA

Question 5

Hybridization probe

Answer 5

Can be DNA or RNA
Bind to specific sequence in DNA
Can be radioactively labelled
Can be used to recognize Restriction Fragment Length Polymorphisms

Question 6

Southern Blot

Answer 6

1. Size-fractionation of complex mixture of DNA using agarose gel-electrophoresis
2. Denaturation of dsDNA by NaOH to break down hydrogen bonds between bases
3. Transfer of ssDNA to rigid support: nitrocellulose filter

Question 7

DNA transfer to nitrocellulose

Answer 7

Capillary action

Question 8

Filter hybridization

Answer 8

1. Single stranded DNA immobilized on nitrocellulose filter
2. Add radioactive hybridization probe
3. Probe anneals to target DNA
4. Wash off probe not hybridized to target DNA
5. Overlay filter with X-ray film for autoradiography

Question 9

Autoradiography

Answer 9

Photographic emulsion film
Chemical process: photons/radiation cause metallic spec to form
Only lanes with probe are visualized

Question 10

Photographic emulsion

Answer 10

Silver halide crystals suspended in gelatine on plastic film
During development, photons/radiation cause spec to form by converting crystals to metallic silver that is visible on film

Question 11

Application of Southern blot and Hybridization technique

Answer 11

1. Diagnosis of genetic disease

Question 12

Sickle-cell anemia

Answer 12

Mutation of beta-globing gene that codes for essential polypeptide in hemoglobin
A to T on 6th codon of beta-globing gene (amino acid from Glu to Val)
Causes aggregation of hemoglobin with low O2
Destroys recognition sequence (CTNAG) for restriction endonuclease Dde1

Question 13

Dde1

Answer 13

Restriction enzyme

Recognizes sequence CTNAG on wild-type beta-globin gene

Question 14

Amniocentesis

Answer 14

Procedure for obtaining fetal cells for genetic testing

1. Sterile needle inserted into abdominal wall into amniotic sac
2. Small amount of amniotic fluid is withdrawn through needle
3. Amniotic fluid contains fetal cells, which are separated from fluid
4. Cells are cultured
5. Cells are tested

Question 15

Testing fetal cells

Answer 15

Chemical analysis
DNA analysis
Chromosomal analysis

Question 16

Chorionic villus sampling

Answer 16

For genetic testing
Can be performed much earlier than amniocentesis
1. Using ultrasound, catheter is inserted through vagina and cervix into uterus
2. Catheter was placed in contact with chorion (outer layer of placenta)
3. Section removes small piece of chorion
4. Cell are used from genetic tests

Question 17

Globin mRNA

Answer 17

make up 80% of mRNA in RBC

Question 18

Heterologous probes

Answer 18

Genes already cloned from related organisms

Question 19

Synthetic DNA probes

Answer 19

Can use reverse genetics to design a DNA probe based on the protein sequence coded by the gene

Question 20

Restriction Fragment Length Polymorphisms

Answer 20

Different restriction sizes of different alleles at chromosome locus
Can be recognized by probes: adhere to sequence of DNA regardless of restriction site location
Can be linked to genetic disorders: irregularities due to recombination