Use of Molecular Genetics in Medicine

Question 1

what is a chromosomal disorder?

Answer 1

defect in whole chromosomes, not just single mistake

Question 2

what are the solutions for doing genetic testing?

Answer 2

enzymes that manipulate DNA
cloning of target DNA into a vector
Polymerase chain reaction (PCR)

Question 3

what is hybridization?

Answer 3

annealing of ssDNA probe to complementary ssDNA

Question 4

what do PCR (primer), sequencing (primer), southern blots, RFLP analysis and allele specific oligonucleotide (ASO probes) use?

Answer 4

DNA

Question 5

what does northern blots use?

Answer 5

RNA

Question 6

what does western blots use?

Answer 6

protein

Question 7

what is restriction endonucleases (class II)?

Answer 7

recognize and cut DNA in a specific and reproducible fashion.
hydrolyze phosphate backbone to give a 5’ phosphate and a 3’ OH
blunt or sticky overhang 4-8 bp in length, 6 bp is most common
homodimers that recognize short, symmetric DNA sequences
Do not have methyl’s activity and thus are very useful for DNA manipulation

Question 8

what is a palindrome?

Answer 8

when read in the 5’ to 3’ direction, the sequence on the “top” strand is identical to that of the “bottom” strand

Question 9

what is the probability of the restriction site occurring in a piece of DNA?

Answer 9

expect that a Hpall recognition site of 4 bp will occur every 256 bp

Question 10

what is DNA polymerase?

Answer 10

enzymes that synthesize a complementary new strand of DNA from DNA or RNA template

Question 11

what are the 4 most used types of DNA polymerase?

Answer 11

DNA polymerase I
Klenow fragment DNA polymerase
Reverse transcriptase
Taq Polymerase

Question 12

what is prokaryotic plasmids?

Answer 12

single, large circular chromosomes - can insert up to 10 kb of foreign DNA

Question 13

what are 3 important feature of cloning vectors?

Answer 13

self replication
must have multiple cloning sites
must have a selectable marker

Question 14

what are the functions of cloning vectors?

Answer 14

replication
sequencing
protein expression

Question 15

what are the founding priniciples of PCR?

Answer 15

DNA polymerase I synthesize in 5’ to 3’
primers - free 3’ OH
heat to denature DNA
exponential increase in the number of DNA molecules

Question 16

what are the components required for PCR?

Answer 16

DNA polymerase
Primers
deoxynucleoside triphosphates (dNTPs)
Magnesium chloride
Buffer
DNA

Question 17

what is a cofactor required for DNA polymerase activity?

Answer 17

magnesium chloride

Question 18

what are the steps in PCR?

Answer 18

denaturation
annealing
primer extension
REPEAT 28-35 times!

Question 19

in gel electrophoresis, what do the nucleic acids migrate towards and why?

Answer 19

towards the cathode, nucleic acids have a negative charge from phosphate backbone

Question 20

what is the difference between northern blots and southern blots?

Answer 20

norther blots contain isloated mRNA separated by gel electrophoresis

Question 21

why do we sometimes call plasmids vectors?

Answer 21

vectors carry DNA of interest

Question 22

what do we need for easy primer design?

Answer 22

information from the sense strand - then we can make both forward and reverse primers from that

Question 23

what is the forward primer complementary to?

Answer 23

antisense strand

Question 24

what makes primer design so difficult?

Answer 24

you want to get primers that work together
anneal and melt at the same temperatures
depends on GC primers (3 hydrogen bonds)

Question 25

how do we amplify mRNA?

Answer 25

we need ds species to PCR and clone | use reverse transcrpitase to take RNA and convert it to DNa

Question 26

in gel electrophoresis, which molecules move faster and slower?

Answer 26

``` fast = small slower = large ```

Question 27

what was the first diagnostic technique devides?

Answer 27

southern blots

Question 28

what does southern blot tell us?

Answer 28

if one or both alleles are different from normal | if we have made or broken a restriction site

Question 29

what does western blot tell us?

Answer 29

if there is a change in size of the protein of interest | use antibody specific to protein of interest

Question 30

what do DNA and RNA techniques use?

Answer 30

DNA probe

Question 31

what does protein technique use?

Answer 31

antibody probe

Question 32

what did Freddy Sanger do and how?

Answer 32

developed the human genome project | took 3' OH off the ribose

Question 33

what technique is commonly use today?

Answer 33

sequencing

Question 34

what occurs in sequencing?

Answer 34

one lane on a gel, products of each reaction will emit a different color when it excited by light

Question 35

what can a single base pair change in RFLP analysis?

Answer 35

change the creation or destruction of a restriction site destroys Sca1 restriction site First: amplify 186 bp frag of MSUD gene Next: digest with Sca1 and see on agarose gel

Question 36

what is used to detect polymorphins or common genetic mutations (CFTR, sickle cells, others)?

Answer 36

ASO (allel specific oligonucleotide) probes

Question 37

what can allele specific PCR detect?

Answer 37

single nucleotide changes

Question 38

what ensures that the PCR is working properly?

Answer 38

internal controls

Question 39

what does sickle cell mutation of beta-globin gene destroy?

Answer 39

Ddel site

Question 40

what does sickle cell mutation of beta-globin gene destroy?

Answer 40

Ddel site