12P. Molecular genetics

Question 1

Types of molecular genetic methods

Answer 1

1) “Cut and paste” (restriction endonucleases+ligases)
2) Visualize (electrophoresis)
3) Hybridization (e.g FISH)
4) Amplify (PCR)

Question 2

Restriction enzymes

Answer 2

Restriction endonucleases
- Cut DNA at specific (palindromic) sequences
Reverse transcriptase (?)

Question 3

Recombinant DNA

Answer 3

2 molecules - Digestion with endonuclease -> sticky ends -> sealed by DNA ligase -> recombinant DNA

• Can be used for gene cloning by the help of bacteria
• Recombinant drugs (e.g vaccines - Ag, hormones etc)
• Therapeutic purpose
• Generation of transgenic (overexpressed gene) reasearch organisms

Question 4

Usage of recombinant DNA technology: differences between them

Answer 4

k

Question 5

Usage of recombinant DNA technology: genotyping

Answer 5

k

Question 6

RFLP

Answer 6

k

Question 7

Causes (SNP, VNTR, InDel) RFLP

Answer 7

k

Question 8

Detection of RFLP by Southern blot: separation of DNA fragments by gel electrophoresis or by capillary gelelectrophoresis

Answer 8

k

Question 9

Detection of RFLP by Southern blot: non-specific staining of DNA (EtBr).

Answer 9

k

Question 10

RFLP (?): Usage of probes

Answer 10

k

Question 11

Detection of RFLP by PCR

Answer 11

k

Question 12

Significance and applications of real-time PCR

Answer 12

k

Question 13

Multiplex PCR

Answer 13

k

Question 14

Significance and detection of VNTR

Answer 14

k

Question 15

VNTR and trinucleotid diseases

Answer 15

k

Question 16

Significance of polymorphism detection in forensic medicine

Answer 16

k

Question 17

DNA fingerprint

Answer 17

k

Question 18

Foreign DNA (bacterial, viral) detection

Answer 18

k

Question 19

Sequencing in genotyping

Answer 19

k

Question 20

NGS

Answer 20

k

Question 21

Study of gene expression methods

Answer 21

1) Northern blot
2) RT (reverse transcriptase)-PCR
3) FISH

Question 22

Northern blot

Answer 22

Study of gene expression - check RNA

- Probe: labelled DNA

Question 23

RT (reverse transcriptase)-PCR

Answer 23

Study of gene expression

- Reproduction of RNA viruses: Reverse transcriptase

Question 24

FISH

Answer 24

Study of gene expression

Question 25

Study of genome: Applications, significance of genetic tests.

Answer 25

k

Question 26

Genetic diagnosis for Fragile X syndrome

Answer 26

Southern blot (PCR for screening)

Question 27

Gel electrophoresis + capillary (gel) electrophoresis

Answer 27

Gel electrophoresis

• Separated according to charge & size (smaller move faster)
• Agarose concentration in gel -> pore size

Capillary (gel) electrophoresis

• Many samples at same time
• Main advantage: different gel matrix can be used -> different separation (RNA, DNA, protein)
• Can separate very small DNA sequences

Question 28

Hybridization

Answer 28

• Hybridization of complementary strands
• DNA-DNA
• DNA-RNA
• RNA-RNA
• Example: FISH

Question 29

Southern blot method + applications

Answer 29

Check DNA
- Electrophoresis -> nitrocellulose filter -> hybridize w/labelled probes (DNA) -> X-ray => Autoradiogram

Applications:

• Detection Fragile X
• Identification of methylation sites