10. DNA Tech + Applications

Question 1

Polymer Chain Reaction (PCR)

Answer 1

Kary Mullis
- in vitro
- DNA amplification
- done in a thermocycler ( to program times, cycles and temps)

Question 2

Steps in PCR

Answer 2

1. DNA denaturation (1-2 mins)
   - heating the reaction mixture
   - double helix to unwind by breaking the hydrogen bonds
2. Primer annealing (1-2 mins)
   - reduce the mixture temperature
   - allows the primers to anneal (relax it to make it easier to work with) to the template DNA
3. Polymerase extension (3-5 mins)
   - raise the temp
   - Taq polymerase to add dNTPS to the growing DNA strand
4. repeat over and over depending on the length of the gene interest

Question 3

Gel electrophoresis

Answer 3

• used to visualize PCR DNA fragment lengths
• separates the DNA fragments by their base pair lengths
• longer fragments are heavier and move slower than smaller

Question 4

Dideoxynucleotide DNA sequencing (Sanger method)

Answer 4

• In vitro DNA synthesis to determine the nucleotide sequence of a DNA fragment
• 4 synthesis reactions (A,T,G,C)
• Closely resembles PCR: genomic DNA, amplified to high concentrations, reaction mix, has ddNTPS

Question 5

What are ddNTPs?

Answer 5

Dideoxynucleotides that lack 2 oxygen atoms, has H rather than oH groups on the 2’ and 3’ carbons
- the lack prevents ddNTP from forming phosphodiester bonds to terminate the growing DNA strand producing fragments that are 1 nucleotide longer than the other

Question 6

In ddNTP DNA sequencing, what number does the primer start on? *see topic 10 page 12

Answer 6

18

Question 7

Which direction are nucleotides synthesized? in ddNTP?

Answer 7

5’-3’ for all, primer is on the 5’ end,

Question 8

TRY: Topic 10, page 14

Answer 8

woot woot

Question 9

Next generation DNA sequencing

Answer 9

• 2001
• fast and cheap
• can produce 50 human genomes per day for $1000 per genome
• utilizes computation power to put fragments together into a genome-wide consensus sequence
• illumina sequencer

Question 10

third generation DNA sequencing

Answer 10

• simialr and parallel process to NGS
• used in genome wide association studies

Question 11

Steps in NGS

Answer 11

1. DNA is fragmented and tagged with adapter molecules attached to both ends of the strands
2. DNA is denatured
3. the adaptor molecules anchor the strand, DNA fragments are placed in a flow cell and amplified to produce clusters of identical strands
4. Laser excites the fluorescent dNTP and a photoreceptor records the wavelength intensity
5. software records the info and converts wavelength to a known dNTP
   NGS determines sequenced by synthesis rather than chain termination

Question 12

Restriction enzymes

Answer 12

• form of recombinant DNA technology
• amplifies, maintains and manipulates specific DNA sequences inside the cell

Question 13

Restriction mapping

Answer 13

• type of restriction enzyme
• uses specific enzymes to cut DNA at specific points resultin in double-stranded breaks
• cheaper than DNA sequencing
• Can obtain start and end sequences, compare the DNA fragments across multiple organism, can be used to insert cloned DNA or PCR products

Question 14

Steps in restriction mapping

Answer 14

1. cut DNA with restriction enzymes - to get fragments of various lengths
2. separate fragments with electrophoresis
3. visualize DNA using a stain or probe

Question 15

Molecular cloning

Answer 15

• a technique that inserts restriction fragments into a vector (carrier fragment of DNA)
• contains the genes to replicate DNA in the cell
  1. vector is inserted into an organism
  2. this amplifies the recombinant DNA molecule within the cell, making DNA clones
  3. isolate DNA products for DNA sequencing and other downstream methodologies

Question 16

DNA fingerprinting/profiling

Answer 16

• uses short tandem repeats (STR0
  aka short repeated DNA sequences (<10bps long)

Question 17

Short tandem repeats

Answer 17

• Inheritance of STR is codominant
• similar to VNTRS
• first examined by PCR to amplify the target STRs
• each allele within the STR produces fragments of distinctive lengths
• can compare the number of repeats and corresponding bands to get a DNA profile using gel electrophoresis

Question 18

Combined DNA index system criteria

Answer 18

1. Must have a known chromosome location that independently assorts from all the other CODIS STRS
   - usually in non-coding regions of the genome
2. Must have multiple alleles per STR locus in all populations examined
   - None of the alleles can be more frequent than 20-25%
3. STR alleles must be consistently, reliably, and accurately able to be amplified via pcr
   - reducing contamination
   - only certain people can do it
4. PCR products must distinguish alleles from each other clearly enough for the automated PCR AMPLIFICATION AND GEL ELECTROPHORESIS TO RELIABLY ID EACH allele

Question 19

Ex) DNA Index System: D1S1656

Answer 19

D = DNA
1 = chromosome number
S = single, only found once in the genome
1656 = position on the chromosome

Question 20

TRY: Topic 10, page 34

Answer 20

woot

Question 21

Paternity index (PI)

Answer 21

X/Y
X = prob. came from father
Y = prob. came from another man
PI > 1 = alleles came from father
P1 < 1 = alleles came from someone else

Question 22

Combined Paternity Index (CPI)

Answer 22

adding up the PI’s for each gene analyzed
CPA > 100 = 99% likely that he is the father

Question 23

How is individual identification done?

Answer 23

1. take dna from deceased
2. compare to DNA database or DNA from a family member

Question 24

What are the 3 kinds of DNA used to identify

Answer 24

1. Y chromy for males
2. Autosomal chromosomes
3. mitochondrial chromosomes for maternal DNA