Molecular Genetic Techniques 1 (L5)

Question 1

modes of inheritance

Answer 1

dominant, recessive, X-linked, mitochondrial

Question 2

relationship b/w genotype and phenotype

Answer 2

genotype determines phenotype

Question 3

two different modes of cell division

Answer 3

mitosis and meiosis

Question 4

markers (biomarkers)

Answer 4

polymorphisms on DNA that are co-segregated w/ the disease phenotype, which presumably are located very close to the disease-causing gene

Question 5

haplotype

Answer 5

combo of many polymorphisms along one chromosome

Question 6

what is recombination efficiency dependent on?

Answer 6

distance from a polymorphism or mutation

Question 7

what is the overall purpose of linkage analysis?

Answer 7

use landmarks that we know on a chromosome to determine where the disease-causing mutation is

Question 8

alleles

Answer 8

different forms of the same gene

Question 9

what is a technique to analyze markers on DNA?

Answer 9

agarose gel electrophoresis

Question 10

agarose gel electrophoresis

Answer 10

agarose creates 3D sieve to analyze biomolecules - DNA travels through pores based on size (larger travel slower, smaller travel faster) -> incubate with fluorescent dye to visualize bands

Question 11

restriction enzymes

Answer 11

proteins from bacteria that attack bacteriophages and chop them into smaller pieces

Question 12

what do restriction enzymes recognize?

Answer 12

palindromic sequences in DNA

Question 13

what type of cuts do restriction enzymes make?

Answer 13

sticky end cuts - leave some ssDNA out

blunt end cuts

Question 14

RFLP

Answer 14

restriction fragment length polymorphism - mutation eliminates a restriction site -> change palindromic sequence -> restriction enzyme no longer recognizes it -> no cut

Question 15

southern blot

Answer 15

1. cleave DNA with restriction enzymes
2. run on agarose gel
3. transfer DNA to nitrocellulose membrane in alkaline solution
4. stain by radioactive probe
5. visualize bands

Question 16

what does alkaline solution do to DNA?

Answer 16

denatures it

Question 17

probe

Answer 17

antisense sequence w/ radioactivity or dye for visualization

Question 18

microsatellites

Answer 18

short tandom repeats of DNA sequences - can have di-nt or tri-nt repeats

Question 19

SNP

Answer 19

single point mutation

Question 20

polymorphism vs. mutation

Answer 20

polymorphism: neutral, mostly doesn’t cause harm
mutation: affects body fxn, changes codon or how gene is expressed - can cause a disease state

Question 21

what are three types of polymorphisms DNA can have?

Answer 21

1. RFLP
2. microsatellite
3. SNP

Question 22

genetic distance

Answer 22

how close a marker is to the disease gene - measured in centimorgans (cM) - can do this to calculate how far apart marker is from disease gene, then use probability fxn to determine how likely is it to find a disease gene at a particular location

Question 23

what does 1 cM correspond to in bp?

Answer 23

1 million bp

Question 24

relationship b/w genetic distance and chance of recombination

Answer 24

smaller the distance, smaller chance of recombination - if very closely linked, most often get parental genotype

Question 25

what can you use to determine how much the disease gene is linked to the marker?

Answer 25

percents of non-recombinant and recombinant gametes

Question 26

family-based linkage analysis

Answer 26

start with a family -> use polymorphic marker -> go through recombinations -> track where it goes

Question 27

what are other applications of DNA polymerisms in medicine?

Answer 27

paternity tests and forensic analysis - criminal identification

Question 28

how to pinpoint the mutated gene?

Answer 28

cytogenetic map -> linkage map -> physical map -> sequence map

Question 29

genetic vs. physical map

Answer 29

genetic: chromosome banding pattern FISH physical: hybridization to plasmid clones

Question 30

sanger dideoxy sequencing

Answer 30

1. Add a primer to the DNA 2. Add DNA pol + dATP, dGTP, dCTP, dTTP 3. Add ddNTP in low concentration 4. ddNTP gets incorporated occasionally 5. when ddNTP incorporated, DNA pol can't extend further 6. get multiple lengths depending on where the ddNTP binds 7. repeat sequence with all four ddNTPs w/ fluorescent colors 8. analyze w/ PAGE

Question 31

why does a dideoxy nt stop DNA polymerase?

Answer 31

it has no 3' OH, which is necessary to extend DNA

Question 32

PAGE

Answer 32

polyacrylamide gel electrophoresis -each band is one nt from last one -each band has fluorescent color read sequentially -> get DNA sequence

Question 33

how can you use sequencing to find a mutation?

Answer 33

compare patient to control electropherogram - differences indicate mutation

Question 34

PCR

Answer 34

cycles of denature, anneal, extend

Question 35

what ingredients are necessary for PCR?

Answer 35

- target DNA - DNA pol - dNTPs - MgCl2 - buffer

Question 36

applications of PCR

Answer 36

1. diagnosis | 2. mutation analysis

Question 37

what increase in DNA does 20 cycles of PCR correspond to?

Answer 37

1 million fold increase in DNA