Molecular Techniques I: DNA

Question 1

Types of dna techniques

Answer 1

“to clone Paul C.Rxn from down South, go to the Library and return with the Sequence”

Cloning

PCR

Southern Blot

DNA library

Sequencing

Question 2

Requirements for cloning

Answer 2

Plasmid

Eukaryotic DNA

Restriction enzymes (endonuclease)

Ligase

Question 3

Cloning procedure

Answer 3

Question 4

Plasmid requirements

Answer 4

Origin of replication

Selectable marker

Restriction site

Question 5

Gimme: what do you use for foreign dna that’s more than 20kb?

Answer 5

Bacterial Artificial Chromosome

Yeast Artificial Chromosome

Question 6

Clustered restriction sites are called

Answer 6

Multi-cloning regions

Question 7

How do restriction enzymes work?

Answer 7

Cut at 4-6bp sequences (aka restriction sites) that are palindromic

Question 8

Possible ligation products after cloning

Answer 8

Question 9

Transformation requirements

Answer 9

Competent cells (electrocompetent or heat-shock based)

Electroporation/Heat shock

Antibiotic selection

Colony screening

Question 10

Transformation procedure

Answer 10

Question 11

Colony screening types

Answer 11

Blue white screening

Hybridization

(Colony PCR w/ flanking primers and gel)

Question 12

Blue white screening procedure

Answer 12

Question 13

Hybridization procedure for colony screening

Answer 13

Question 14

How do you make a DNA/RNA probe?

Answer 14

Find 6-7 aas in a row in a region with the least degeneracy

Total probe size = 18-21 nucleotides long

Radiolabel 5’ end

Question 15

Polymerase chain reaction procedure

Answer 15

Question 16

Calculation of copy number after PCR

Answer 16

2^n where n = # ds strands generated/cycle number +1

Question 17

Describe how you would use PCR to identify mutations in known genes using the CFTR gene as an example

Answer 17

(check sheet for answer)

Need to design primers that flank the normal region of the gene and the deleted/mutated region of the gene, amplify DNA then run a gel

If HM normal, gel will show ONE large band

if HT, gel will show 2 bands (one large, one small)

if HM mutant, gel will show ONE small band (mutated region)

Question 18

Southern Blotting procedure

Answer 18

Question 19

SNP-based Restriction fragment length polymorphism analysis

Answer 19

SNP abolishes restriction enzyme site

Question 20

STR based RLFP analysis concept

Answer 20

Question 21

DNA library types

cDNA library construction process

Answer 21

Genomic and cdna

Question 22

Sanger sequencing (old school) concept and procedure

Answer 22

Based on chain termination (via ddNTPS) and 4 separate reaction mixtures

Question 23

Sanger sequencing (new school) procedure

Answer 23

(still chain termination) One reaction and capillary gel. Signal read by laser since ddNTPs have flourophore

Question 24

Next Generation sequencing procedure

Why is it called “massively parallel?”

Answer 24

Question 25

Ion torrent Next Gen Platform

Answer 25

Question 26

Reverse termination/Bridge PCR (Illumina) NExt Gen

Answer 26

Question 27

Bridge PCR for Illumina

Answer 27