DNA Techniques- Lecture 8/19/21

Question 1

Restriction Endonuclease

Answer 1

Cleaves within the DNA at a particular sequence (palindromic sites)

Question 2

Gel electrophoresis

Answer 2

When current is applied DNA runs to the positive side, bigger molecules move faster than small

Question 3

Pulsed field gel electrophoresis

Answer 3

Used to separate big pieces of DNA, including chromosomes

Question 4

Southern blot

Answer 4

Cleave DNA with restriction endonuclease->run on gel->transfer to nitrocellulose->Hybridize with labeled probe for gene of interest

Question 5

Detecting trinucleotide expansion with southern blot

Answer 5

Larger the fragment, more trinucleotide expansions

Question 6

Detecting point mutation using southern blot

Answer 6

If mutation is in cleavage site, will lead to abnormal fragment lengths

Question 7

RFLP

Answer 7

Restriction fragment length polymorphism- when there is some polymorphism in the human genome that adds or takes away a cleavage site

Question 8

FISH

Answer 8

Fluorescent in situ hybridization-> denature chromosomes and at fluorescent probe

Question 9

Northern blots

Answer 9

Similar to southern blots, but with RNA

Question 10

Western blots

Answer 10

Look for protein-> uses labeled antibodies to see if protein of interest is present

Question 11

DNA band shifts

Answer 11

When a protein is interacting with a piece of DNA, it becomes larger and moves slower through the gel

Question 12

DNA footprinting

Answer 12

When looking for a specific binding site, add DNase which nicks the phosphodiester bond, where there is a “shield” there is a protein binding site

Question 13

Thermocycler

Answer 13

Machine used in PCR to heat up and cool off DNA to denature and re-anneal DNA

Question 14

Primers

Answer 14

Short ~20 bop oligonucleotides that bind in both forward and reverse direction to the gene of interest to amplify fragments

Question 15

Detecting expansion through PCR

Answer 15

PCR not viable for genes ~5,000 by and over because the polymerase falls off the DNA, will not get results

Question 16

RT-PCR

Answer 16

Rat stands for reverse transcriptase, used to look for RNA in the sample by turning it into cDNA via reverse transcriptase

Question 17

Oncotype score

Answer 17

Using RT-PCR, used to detect cancer genes in patients to give them a recurrence score, about 16 genes of interest

Question 18

Molecular cloning

Answer 18

Cleave gene and plasmid with restriction enzyme, gene incorporates into plasmid via sticky ends, kept in library by bacteria

Question 19

Sticky ends

Answer 19

EcoR1 makes “sticky ends” by creating complimentary base pairings because of the palindromic sequence

Question 20

genomic library

Answer 20

Constructed directly from fragments of genomic DNA

Question 21

CDNA library

Answer 21

Derived from mRNA, converted to DNA by reverse transcriptase first

Question 22

Dideoxy analogue

Answer 22

Uses a dideoxy sugar which lacks 3’ hydroxyl to terminate chain elongation

Question 23

Sanger method

Answer 23

Uses dideoxy sugars to randomly stop at a given location, know the opposite basepair is the opposite