gene technologies

Question 1

proteome =

Answer 1

the full range of proteins a cell can prod

Question 2

genome =

Answer 2

all of the DNA in a cell/organism

Question 3

sequencing a genome requires…

Answer 3

working out the DNA base seq for all the DNA in a cell

Question 4

the human genome project

Answer 4

took 13 years –> completed in 2003

Question 5

why is it easier to use the genome of simpler organisms to identify potential antigens for vaccines?

Answer 5

• they dont contain introns but eukaryotes do contain introns and regulatory genes (alternative splicing occurs)
• the genome can be directly used to obtain the proteome of the organism –> this can be used to identify potential antigens

Question 6

examples of recombinant DNA technologies include…

Answer 6

1. creating DNA fragments for in vitro/in vivo cloning
2. genetic fingerprinting
3. genetic screening, counselling & location of specific genes

Question 7

the 3 ways of prod. DNA fragments

Answer 7

1. restriction endonucleases
2. reverse transcriptase
3. gene machine

Question 8

recombinant DNA technology =

Answer 8

the combining of diff organisms’ DNA –> enables scientists to manipulate & alter genes to improve industrial prcesses/medical treatment

Question 9

restriction endonucleases

(1/3 method of prod. DNA)

Answer 9

naturally occur in bacteria as defense mechanism
1. active sit of endonucleases comple. to specific DNA base seqs –> recognition seqs
2. therefore, each enzyme cuts the DNA at a specific location
3. some endonucleases cut at same location of double strand to create blunt end, some endonucleases cut to create staggered ends & exposed bases
4. staggered ends = ‘sticky ends’ –> palindromic –> have ability to join to DNA with comple. base pairs

Question 10

reverse transcription

(2/3 method of prod. DNA)

Answer 10

1. isolate the mRNA transcribed for the desired gene
2. the reverse transcriptase joins the comple nucleotides to the mRNA seq.
3. single-stranded ‘cDNA’ is prod.
4. the DNA polymerase joins nucleotides to the cDNA to prod double-stranded DNA

Question 11

why is the cDNA intron-free?

Answer 11

it’s based on the mRNA template

Question 12

gene machine

(3/3 method of prod. DNA)

Answer 12

• uses computerised machine
• a.a seq of protein used to determine DNA seq
• PCR amplifies amount of oligonucleotides (which make up gene)
• DNA fragments prod = intron-free –> can be transcribed in prokaryotic cells

Question 13

why is use of a gene machine faster than reverse transcription to prod DNA. fragments?

Answer 13

reverse transcription has slow enzyme-controlled reactions

Question 14

name the 2 methods of amplifying DNA fragments to obtain a large amount

Answer 14

1. in vivo cloning
2. in vitro cloning

Question 15

in vivo cloning

Answer 15

1. restriction endonucleases cut at recognition sites, leaving ‘sticky ends’
2. promotor and terminator regions added –> RNA polymerase can attach and detach –> transcription starts & stops
3. insert DNA into vector –> plasmid cut using same res. endo. –> comple ‘sticky ends’ -> ligase enzyme anneals DNA & vector –> DNA ligase forms phoshodiester bond between adj. nucleotides on sticky ends
4. vector inserted into host cell –> cell membrane of host cell needs increased permeability –> mixed w Ca^2+ & heat shocked
5. addition of marker genes

Question 16

issues w recombinant plasmids

(which makes identification important)

Answer 16

1. recombinant plasmid not inside cell
2. plasmid re-joins before DNA frag enters
3. DNA frag sticks to itself (instead of plasmid)
   marker genes used to identify which genes successfully took up the recombinant plasmid

Question 17

in vitro cloning

Answer 17

uses ‘polymerase chain reaction’ in a thermocycler

Question 18

describe and explain how PCR is used to amplify a DNA fragment (4)

Answer 18

1. thermocycler contains DNA fragment, DNA polymerase, nucleotides & primers
2. heat to 95 degrees to break H-bonds –> splits DNA into single strands
3. reduce temp to 55 degrees so primers anneal/bind to DNA
4. increase temp to 72 degrees so DNA polymerase joins free comple nucleotides to exposed DNA strand

Question 19

in every PCR cycle, how does the amount of DNA increase?

Answer 19

exponential increase –> doubles each cycle

Question 20

advantages of PCR

Answer 20

1. automated –> more efficient
2. rapid –> billions of copies within hrs
3. doesn’t req living cells

Question 21

what is a DNA probe? (2)

Answer 21

1. short single-strand of DNA
2. with bases comple to the DNA/gene

Question 22

why are DNA probes used?

Answer 22

• fluorescent/radioactive label on probe allows identification of specific base seqs of DNA

Question 23

DNA hybridisation (used for medical diagnosis)

(as a result of use of DNA probes and identifying a particular allele)

Answer 23

1. DNA heated –> double strand split into single strands
2. split DNA mixed w comple single-stranded DNA
3. strands will anneal

Question 24

genetic fingerprinting =

Answer 24

analysis of VNTR DNA fragments

Question 25

general process of genetic fingerprinting

Answer 25

1. *extraction* of DNA --> from blood, saliva etc (PCR used if sample too small) 2. *digestion* --> **restriction endonucleases** cut DNA into fragments --> then VNTRs added 3. *separation* --> DNA samples loaded into small wells in agar gel placed in buffer liq w voltage applied --> -vely charged DNA moves towards +ve end of gel --> **gel electrophoresis** 4. *hybridisation* --> DNA probes **comple** to VNTRs --> bind 5. *development* --> VNTRs & probes transferred to nylon sheet --> **x-ray film**/uv light used to show probe 6. *analysis*

Question 26

genetic fingerprinting mark scheme ans

Answer 26

1. extract DNA & add **restriction endonucleases** 2. seperate fragments using gel electrophoresis 3. treat DNA to **expose** bases 4. probe will bind/hybridise to gene 5. use **autoradiography**/x-ray film to show the bound probe

Question 27

gel electrophoresis

Question 28

analysis of genetic fingerprinting

Answer 28

* position of DNA bands (VNTRs) used to compare genetic relationships