DNA Profiling

Question 1

What is a genome?

Answer 1

-all of the genetic material that it contains

Question 2

What are genes?

Answer 2

-regions of DNA on chromosomes that code for polypeptides

Question 3

What are introns?

Answer 3

-larger, non coding regions of DNA in between the genes

Question 4

What is a tandem repeat?

Answer 4

Repetitive sequences of DNA that do not code for proteins found within introns

Question 5

What is a variable number tandem repeat (VNTR)

Answer 5

-A location in a genome where a short nucleotide sequence is organised as a tandem repeat
-these can be found on many chromosomes, and often show variations in length (number of repeats) among individuals
-they are 10-100 base pairs long, repeated 50 to several hundred times

Question 6

What are short tandem repeats?

Answer 6

-smaller repeated sequences within the non-coding DNA
-they are 2-9 base pairs long and are repeated 5 to 15 times

Question 7

Why does the number of short tandem repeats vary between individuals?

Answer 7

-because we inherit different lengths of repeats from each parent
-it is these different patterns that is used in DNA profiling

Question 8

Explain the first step of the method for DNA profiling

Answer 8

1) Extract the DNA
-from saliva, blood drop, hair, semen, bone sample (only tiny amounts are needed because lab techniques can copy the DNA many times to give a large sample to analyse)
-then the DNA needs to be purified because the histone proteins need to be removed using protease enzymes before the DNA is sequenced

Question 9

Explain the second step in the method for DNA profiling

Answer 9

2) Digest the sample with enzymes
-the DNA sample is cut into smaller fragments using restriction enzymes
-they are always cut at specific base pairings because the shape of the enzymes active site will be complementary to the shape of a particular sequence of bases in the DNA, so an enzyme substrate complex can form and the relevant bonds are broken

Question 10

Explain why restriction enzymes come from bacteria?

Answer 10

Bacteria have them as it stops viral DNA taking over the cell as it can be broken down into little pieces helping the cell survive

Question 11

Explain the third step in the method for DNA profiling.

Answer 11

3) Separation of the DNA fragments: Gel electrophoresis
-the fragments need to be separated in order to look for patterns that can then be analysed, done through a technique called Electrophoresis

-DNA fragments are put into a ‘well’ in a block of gel, along with some loading dye to make the DNA visible
-this is placed in an alkaline buffer solution, which regulates pH and helps to carry the electrical charge across the gel
-the gel then has an electric current pass through it
-the DNA fragments move towards to positive end (because phosphate groups in DNA give a negative charge)
-the smaller the fragment, the further it moves as has less resistance to gel
-it is important to let it run for sufficient time to allow all the DNA fragments to separate out fully

The DNA is then made single stranded ready for analysis
-a nylon membrane is then placed on top of the gel, with absorbent paper on top. This draws the top strands of DNA onto the nylon = Southern blotting
-the DNA strands are fixed in place onto the nylon membrane by either UV light or heat at 85 degrees

Question 12

Explain the fourth step in the method for DNA profiling

Answer 12

4) Hybridisation
-now DNA probes are added to look for particular mini/micro satellites in the DNA
-the probe has a complementary base sequence to the sequence you are looking for
-when they attach = hybridisation
-the probes are usually used to locate the microsatellites in the DNA because these are more variable from person to person
-any excess probes are washed away

Question 13

What is a probe?

Answer 13

-stretches of single stranded DNA used to detect the presence of complementary nucleic acid sequences by hybridisation
-they are usually labelled with radioisotopes or fluorophores to enable their detection
-they are 100- 1000 base pairs long

Question 14

Explain the fifth step in the method for DNA profiling

Answer 14

5) making it visible
-we cannot see DNA, but if the probes used are radioactive or fluorescent they can be detected wherever they have attached
-if radioactive probes are used placing the nylon membrane onto an X-ray film causes the film to fog where the radiation is
-if fluorescent probes were used, place the nylon membrane under UV light and the probes will glow
-usually at least 13 different STR’s will be looked for when doing a DNA profile. This ensures the wrong person isn’t found as the chances of having 12 different STR’s is hundreds of millions to one

Question 15

What is the PCR (Polymerase chain reaction) used for?

Answer 15

Making multiple copies of the DNA sample so there is enough to analyse

Question 16

What is needed for the PCR technique?

Answer 16

-original tiny DNA sample
-excess of free nucleotides with the 4 different bases
-primers (short pieces of single-stranded DNA which start the copying process) 12-15 nucleotides long
-DNA polymerase enzyme (Taq polymerase as comes from thermophilic hot-spring bacteria so not denatured by high temperatures in the machine)

-all of these are mixed together in a small tube and placed into the PCR machine

Question 17

What is the first step for PCR technique? Include temperature needed

Answer 17

-temperature inside is 90-95 degrees for 30 seconds
-this breaks the hydrogen bonds between the 2 strands of DNA, separating them

Question 18

What is the second step for PCR technique? Include temperature needed

Answer 18

-temperature decreased to 55-60 degrees
-primers join to both ends of the single stranded DNA (short single stranded section of DNA complementary to the ends of the strands needed for replication to start)

Question 19

What is the third step for PCR technique? Include temperature needed

Answer 19

-temperature increased to 70-75 degrees for 1 minute
-this is the optimum temperature for the chosen DNA polymerase enzyme (Taq polymerase) to work
-this adds bases to the primers, extending the complementary strands
-this results In double stranded DNA genetically identical to the original sample

Question 20

Why might you not get as many copies of the DNA made as expected?

Answer 20

-may not be enough primers present
-primers might not attach to all of the DNA strands
-may be insufficient nucleotides available
-2 separated DNA strands may just rejoin to each other rather than the primers

Question 21

What is a similarity and difference between probes and primers?

Answer 21

-similarity = both single stranded sections of DNA
-difference =
probes are used to locate a particular base sequence + 100 to 1000 nucleotides long
Primers are used to start DNA replication of the DNA strands in PCR and are 15 to 25 nucleotides long