Manipulating genomes

Question 1

what does human genome contain

Answer 1

2% Exons which codes for proteins
98% Introns which are involved in gene expression

Question 2

what do Introns contain

Answer 2

Satellite DNA

Question 3

2 different types of satellites DNA

Answer 3

mini satellite (VNTRS) 20-50 bases long repeat 50-100 times
micro satellite (STRs) 2-4 bases long repeated 5-15 times

Question 4

use of DNA profiling

Answer 4

used in criminal investigations to identify suspencts

used in forensic medicine to indenture bodies or body parts that are unidentifiable e.g from a bomb blast

determine familial relationships

Question 5

7 steps of DNA profiling

Answer 5

1. DNA is isolated from collected sample
2. PCR is used to amplify DNA sample
3. use restriction endonuclease to cut the amplified DNA molecule into fragments
4. Separate the fragments using gel electrophoresis
5. Southern blotting
6. hybridisation- add radioactive or fluorescent probes that are complementary and bind to specific VNTR regions
7. analyse the evidence

Question 6

requirements of PCR

Answer 6

1. Target DNA or RNA being amplified
2. primers(forward and reverse) they are short sequences of single stranded DNA that have sequenced complementary to the 3’ end of the DNA being copied
3. DNA polymerase used to build new DNA strand. Taq polymerase
4. free nucleotides
5. buffer solution to provide optimum pH

Question 7

where does PCR occur

Answer 7

thermal cycler equipment

Question 8

steps for PCR

Answer 8

1. Denaturation: the double stranded DNA is heated to 95 degrees which breaks the hydrogen bond between the two DNA strands
2. Annealing: the temperature is decreased to 50-60 degrees so that primers can anneal ti ends of the single strands of DNA
3. Elongation/ extension: temperature is increased to 72 degrees and it’s optimum temperature for taq polymerase. free nucleotides pair up to exposed bases by complementary base pairing and taq polymerase attach the nucleotides together forming 2 new DNA fragments

cycle starts again and each new cycle doubles the amount of DNA so 30 cycles is 2^30 is around 1 billion new DNA

Question 9

process of DNA sequencing

Answer 9

DNA for sequencing is mixed with a primer, DNA polymerase and an excess of free nucleotide and terminator bases

the mixture is placed in a PCR machine

each time a terminator based is incorporated instead or a normal base the synthesis of DNA is terminated. this results in many DnA fragments of different lengths

the DNA fragments are separated by the lengths by gel electrophoresis in capillary tubes. the fluorent tag on the terminator bases are used to identify the final base of each fragments. laser detect colour and thus order of sequence

this shows the sequence of the new strand which can be used to work out original strand