DNA Fingerprinting

Question 1

why is DNA unique in individuals

Answer 1

as the genome in a human has a specific order of nucleotides and they have a unique pattern of polymorphic sequences in his/her chromosomes (exception: twins)

Question 2

what are restriction enzymes +function +example

Answer 2

• they are molecular scissors (enzymes), made by bacteria, that cut at a restriction site in a nucleotide sequences
  example: EcoRl

Question 3

what is DNA’s charge + reason

Answer 3

-negative

Question 4

how and why can DNA fragments move in an electrical field

Answer 4

• due to the negative CHARGE of the DNA, and because opposites attract, there is a positive end at the opposite of the electrical field that pulls the DNA fragments
• the smaller the piece, the further it moves down the electrophoresis gel

Question 5

why do we need to use the same restriction enzymes for a crime scene investigation

Answer 5

• to recognize restriction sites + cut

- the site where they cut generate specifically sized fragments of DNA that are unique to an individual

Question 6

what are polymorphic DNA sequences +effect on restriction enzymes

Answer 6

• regions on human chromosomes that exhibit a great deal of diversity
• they can change the size of the restriction enzymes

Question 7

what are short tandem repeats (satellite DNA)

Answer 7

• they are polymorphic sequences that lie close to each other on the chromosome that repeat for a while
• highly variable between individuals

Question 8

where are STR’s found

Answer 8

in the inter-genie region

Question 9

what are STR’s used for

Answer 9

• forensic identification
• paternity testing
• genetic disease diagnosis

Question 10

how can STR’s be used

Answer 10

• to establish human identity (fingerprinting)

Question 11

what is a restriction site

Answer 11

• a specific sequences of base pairs in DNA

- its a palindromic sequence (read the same way right to left as it is done left to right)Ex

Question 12

what is the restriction site for the enzyme EcoRL

Answer 12

AATT and TTAA

Question 13

how do restriction enzymes cut

Answer 13

• if it cuts at one location, two fragments produced

- if it cuts at two locations, three fragments produced

Question 14

what happens when two pieces of DNA are identical

Answer 14

-a specific restriction enzymes will cleave the two DNA samples in the same way, generating two fragments of the same size

Question 15

what are the steps of DNA profiling

Answer 15

1. DNA is amplified by PCR reaction (polymerase chain reaction)
2. DNA is cut up into short fragments using restriction enzymes
3. Gel Electrophoresis is used to separate fragments by size and charge

Question 16

what do you do during the PCR reaction

Answer 16

1. Denaturation: temperature raised to separate the DNA strands at 90.C
2. Annealing: primers allowed to anneal as temperature drops (50.C)
3. Extension: Tagpolymerase elongates new strands of DNA

Question 17

process of gel electrophoresis lab

Answer 17

• In a gel electrophoresis lab a mixture of restriction enzymes, DNA samples and loading dye (to indicate where the fragments are and to weigh down the mixture) are inserted into their respective wells in an agarose gel (this mixture is measured with a micropipette which inserts indicated amounts into microcentrifuge tubes, the mixture is then mixed and collected to the bottom of the microcentrifuge tube using a pulse centrifuge).
• When the restriction enzymes are inserted into the tubes and once they are all in the microcentrifuge tube rack, they are to be incubated for 45 minutes at 37°C in a water bath so that the enzymes can work under an equal and optimum reaction rate.

Question 18

why is a buffer used in the lab (when gel is submerged in buffer solution)

Answer 18

-optimal PH for lab for the enzymes (stabilize experiment)

Question 19

why do we use a standard solution

Answer 19

• A standard solution, the DNA from the crime scene and the DNA samples of crime suspects are inserted into these wells.
• the standard solution is used for comparative reasons. Once the lab is over and the bands of the DNA solutions appear the standard solution shows bands where the number of base pairs is already known.

Question 20

why water bath

Answer 20

Water bath and consistent temperature regulation used to make the rate of reaction of the restriction enzymes consistent and equal amongst different samples, and use a temperature which works best for the restriction enzyme.