DNA Technology I and II

Question 0

Restriction enzymes

Answer 0

Bacteria protect own DNA by methylating bases because their restriction enzymes attack foreign DNA

Recognize palindromic sequences

Question 1

Nucleic acid hybridization

Answer 1

Annealing of one strand to complement.

Probe usually DNA, template DNA or RNA.

Question 2

Restriction fragment linked polymorphisms (RFLPs)

Answer 2

DNA fragments used to identify sequence differences

Question 3

Gel electrophoresis

Answer 3

Size separation; negative cathode to positive anode; agarose for large nucleic acids, acrylamide for smaller.

Question 4

Southern blotting

Answer 4

From gel electrophoresis to nitrocellulose paper with strong base to transfer and denature DNA, NA immobilized on paper ready to bind with radioactively labelled probes.

Question 5

Cloning

Answer 5

Restriction endonucleases to create sticky ends, isolate fragment via gel and ligate into cloning vector with T4 DNA ligase (bacteriophage enzyme), transform into e. Coli

Question 6

Bacterial plasmid

Answer 6

Vector
F1 plasmid that carries antibiotic resistance genes, many RE cut sites
Carries 15 kb

Question 7

Bacteriophage lambda

Answer 7

Vector
Infect bacteria to produce viral particles, lysis
Carries 20 kb

Question 8

Cosmids

Answer 8

Vector
Essential genes removed, sequences at ends used to replicate and package DNA into viral particles
Carries 45kb

Question 9

Bacterial artificial chromosomes (BACs)

Answer 9

Bacteria fooled into carrying BAC, dsDNA

Carries 100-300kb

Question 10

Yeast artificial chromosomes (YACs)

Answer 10

Linear dsDNA with telomeres and centromeres

Carries 100-2000kb

Question 11

Genomic v cDNA library

Answer 11

Genomic: digest DNA with RE to isolate
cDNA: mRNA library to get expressed genes with introns removed
mRNA enriched with poly A tails, primer for reverse transcriptase, RNA degrades, ssDNA used as template by DNA polymerase to make ds cDNA to clone

Question 12

Polymerase chain reaction

Answer 12

Replicate via primer directed DNA replication using thermophilic DNA polymerase
Amplification is # of starting template x 2^# of cycles

Question 13

Northern blots

Answer 13

Similar to southern blot but for detecting RNA, used purified mRNA

Question 14

Protein electrophoresis

Answer 14

Proteins will only be charged at certain pHs
Make charge irrelevant to separate by size with denaturing protein electrophoresis (heat in detergent sodium dodecyl sulphate, SDS coats proteins with negative charge, b-mercaptoethanol reduces disulfide bonds
Isoelectric focusing: pH gradient gel, protein migrate till they reach a point in which they have no charge
2D gel electrophoresis: IEF and denaturing gel electrophoresis

Question 15

Western assay

Answer 15

Similar to southern but protein on paper is bound to by primary antibodies then secondary antibodies

Question 16

DNA microarrays

Answer 16

DNA probes on glass slides, mRNA samples from cells/tissues fluorescently labelled hybridizes, analyses expression of many genes

Question 17

Cluster assay

Answer 17

Displays microarray data, compares all expression patterns to each other, compare joined patterns to all others

Question 18

RNA interference

Answer 18

DsRNA hybridizes to and degrades mRNA and down regulates genes, protects against RNA viruses
Poor specificity because of homologous genes