Day 3, Lecture 1 (Aug 24) Genetics III: Recombinant DNA Technology and DNA Mutation

Question 1

Medical Applications of DNA Technology

Answer 1

• Identification of disease-causing genes
• Molecular Genetic (DNA) Diagnosis
• Genetic diagnosis of infectious disease (such as Tuberculosis, HIV)
• Synthesis of recombinant proteins for therapies (e.g. insulin, growth hormones)
• Forensic applications (DNA fingerprinting)
• Gene therapy

Question 2

Procedure of DNA cloning

Answer 2

A DNA fragment is inserted into a vector to create a recombinant vector. A vector is a replicon that makes multiple copies of itself (clones) when introduced into a host bacterial cell ( transformation)

Question 3

Polymerase Chain Reaction (PCR)

Answer 3

• In vitro method for amplifying DNA
• Over million-fold amplification in about 2 hours, starting from a small quantity of input DNA
• DNA is used for further analysis/manipulation as with cloned DNA
• Replaced cloning as the preferred method for amplifying DNA

Question 4

Reverse Transcription- Polymerase Chain Reaction (PCR)

Answer 4

Question 5

Medical applications of PCR

Answer 5

• Advantages
  
  • Identification of minute quantities of DNA
  • Detection of specific DNA sequences
• Mutation detection (molecular diagnosis)
• DNA sequencing (molecular diagnosis)
• DNA fingerprinting (identity/paternity testing)
• Diagnosis of infectious diseases (TB, HIV)
• RT-PCR:
  
  • Detection of splice-site mutations convenient mutation detection (no introns)

Question 6

Missense Mutation

Answer 6

• Replaces one a.a. codon for another

Question 7

Nonsense mutatation

Answer 7

Replaces an a.a. codon for a stop codon

Question 8

Splice-site mutation

Answer 8

Creates/destroys splice sites

Question 9

Classification of DNA mutations

Answer 9

Question 10

Question 11

In-frame deletion vs. Out-of-frame deletion

Answer 11

Question 12

Question 13

Premature STOP codons induce ______

Answer 13

• nonsense mediated RNA decay

Question 14

Explain Splice Site mutations

Answer 14

Question 15

Diseases caused by triplet repeat expansion

Answer 15

• Note that sense these are naturally occurring repeats once there is an occurence you can have anticipation and the occurence of a genetic disorder at progressivley earlier age in successive generations is likely
  
  • Disease severity is proportional to the length of the repeat expansion
  • Expanded repeats are unstable and they may increase in length from one generation to the next

Question 16

What is the gold standard for confirmation of mutations

Answer 16

Sanger’s DNA seqencing

Answer 17

Question 18

Answer 18

Question 19

Question 20

Answer 20

Question 21

Question 22

Most mutations arise due to _____

Answer 22

• replication errors

Question 23

Causes of mutations

Answer 23

Question 24

The ___ dinucleotide is a mutation hotspot

Answer 24

• CpG dinucleotide
  
  • mutation hotspot with 8.5 times greater mutation rate over other dinucleotides

Question 25

Nucleotide excision repair

Answer 25

• repair for UV-induced damage

Question 26

Mutator phenotype

Answer 26

Defects in DNA repair pathways cause acculation of multiple mutations

Question 27

____ is a common consequence of defective DNA repair

Answer 27

Cancer

Question 28

DNA Repair mechanisms

Answer 28

Question 29

Unequal recombination causes

Answer 29

• deletions/duplications
  
  • Note flanking of low copy repeats can lead to this miss pairing and thus lead to duplications and deletions

Question 30

Chromosome 15 has an area that is greatly effected by unequal recombination causing deletions/duplications thus leading to Pader Willi Syndrome and Angelman Syndrome. What is this area flanked by?

Answer 30

low copy repeats of the HERC2 gene

Question 31

restriction enzyme

Answer 31

an enzyme produced chiefly by certain bacteria, having the property of cleaving DNA molecules at or near a specific sequence of bases.

Question 32

cloning vector

Answer 32

A cloning vector is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes.

Question 33

Southern blot

Question 34

Northern blot

Question 35

Mutagenesis

Answer 35

is a process by which the genetic information of an organism is changed in a stable manner, resulting in a mutation. It may occur spontaneously in nature, or as a result of exposure to mutagens