Recombinant DNA technology

Question 1

What is the genome

Answer 1

full set of genes in each cell

Question 2

why are sequencing projects important

Answer 2

allows genome comparison between species to determine evolutionary relationships

can identify potential antigens to use in vaccines

comparisons between individuals allows development of personalised medication

Question 3

What is the proteome

Answer 3

full range of proteins that can be coded by the genome

Question 4

How can you determine proteome of simple organisms

Answer 4

as there are no introns in the DNA, the genome can be translated into the proteome, as all genes are coding.

Question 5

What did the human genome project do

Answer 5

determined the sequence of bases in a human genome

Question 6

How can we use the human genome project

Answer 6

screening for mutated sequences allows identification of genetic disorders before symptoms occur

Question 7

How long did the HGP take and how long could it take now

Answer 7

15 years

as little as 26 hours

Question 8

What is a transgenic organism

Answer 8

an organism that has received a DNA transfer

Question 9

how can transferred DNA be translated in transgenic organisms

Answer 9

as genetic code and transcription and translation machinery are universal for all species

Question 10

3 ways of forming and isolating DNA fragments

Answer 10

Reverse transcriptase

restriction endonuclease

gene machine

Question 11

How can you use reverse transcriptase to isolate DNA fragments

Answer 11

Cell that produces target protein is chosen as has lots of mRNA for the protein

Reverse transcriptase’s attach free nucleotides together that are complementary to the mRNA strand

a single strand of cDNA is formed

DNA polymerase makes cDNA double stranded with nucleotides.

(cDNA has no introns)

Question 12

What does reverse transcriptase do and where does it naturally occur

Answer 12

makes DNA copies from mRNA

occurs in retroviruses e.g. HIV

Question 13

What do restriction endonucleases do

Answer 13

They cut DNA at restriction sites to give DNA fragments

they are complementary to the base sequence at specific restriction sites

some cut through at the same location in both strands of DNA to produce a blunt end

some create staggered ends with exposed bases, these are palindromic and called sticky ends as can join comp base pairs

Question 14

How does a gene machine create DNA fragments
ADBOJ

Answer 14

amino acid sequence of specific protein is identified, then the mRNA and DNA sequence from that

sequence entered into computer to pass biosafety and security checks

computer creates small sections of overlapped DNA strands called oligonucleotides

these can join to form DNA sequence of entire gene

Question 15

What does the process of PCR do

Answer 15

Increases the number of DNA fragments

Question 16

Describe the process of PCR
HCPD

Answer 16

DNA fragment heated to 95 degrees so hydrogen bonds break to separate strands

mixture is cooled to 55 degrees and primers are added to anneal to DNA fragment

heat mixture to 72 degrees so DNA polymerase can attach nucleotides to form 2 new double stranded fragments

Question 17

what do primers do in PCR

Answer 17

primers are short sequences of DNA that join DNA fragments so that DNA polymerase has a basis to attach nucleotides together

Question 18

Describe the process of transformation(In Vivo)
DTPLT

Answer 18

DNA cut using restriction endonucleases to create sticky ends

promoter and terminator region are added to fragments for translation

the same restriction endonucleases are used to cut open plasmid to allow complementary regions for fragment to join

DNA ligase is the enzyme used to join fragment into the plasmid

recombinant plasmid transferred into bacteria via heat shock or Ca2+

Question 19

What are the issues with (in vivo) transformation

Answer 19

not all plasmids take up foreign DNA

not all recombinant plasmids will be taken up by bacterial cells

Question 20

How to identify if plasmids have been taken up by bacteria(Marker genes)

Answer 20

Bacteria is grown on agar plates containing antibiotics

if bacteria survives, plasmid has been taken up as plasmids contain antibiotic resistant genes

if bacteria dies, it does not contain the plasmid

recombinant plasmids will die as recombinant gene will interfere with antibiotic resistant gene

Question 21

How to identify if plasmids are recombinant
GFP

Answer 21

DNA fragment inserted into marker gene that encodes fluorescence

plasmids will not fluoresce if recombinant as foreign fragment has been taken up and GFP wont be made

Question 22

What is gene therapy

Answer 22

curing/treating disorders by inserting functional alleles to mask faulty ones

Question 23

Process of gene therapy

Answer 23

healthy allele is isolated and inserted into faulty cells by plasmids

if mutant allele is recessive, a dominant allele must be inserted

if mutant allele is dominant, DNA must be inserted to silence the allele

Question 24

Difference between somatic and germline gene therapy

Answer 24

Somatic is when alleles are altered in body cells

Germline is when alleles are altered in sex cells and are passed onto offspring ( illegal as offspring cannot consent to the therapy )

Question 25

What are DNA probes

Answer 25

Short sections of DNA that are complementary to a known sequence e.g. a mutated allele

labelled with a fluorescent tag or radioactive tag

Question 26

Why are DNA probes used

Answer 26

Genetic screening, to see whether an individual possesses a recessive mutated allele for a certain disorder

or to evaluate risk of developing disease such as cancer

Question 27

How do DNA probes work

Answer 27

labelled DNA probe is mixed with denatured DNA samples from an individual

if individual has mutated allele the probe will bind to comp base sequence in one strand

this hybridised DNA is detected using radiation or fluorescence

Question 28

What is DNA hybridisation

Answer 28

DNA sequences from different species have comp base pairs and will hybridise

Question 29

What is genetic counselling after screening

Answer 29

providing of information and support about results from genetic screening

Question 30

What is genetic fingerprinting

Answer 30

method used to produce a pattern of DNA bands from an individuals genome

Question 31

What are VNTRs and how do they vary between individuals

Answer 31

Variable number tandem repeats

short repeating sequences of DNA in non coding regions

vary in length and number of repeats so probability of 2 people having same vntrs are low

Question 32

How is DNA fingerprinting carried out
AFEPVDC

Answer 32

Extraction of certain DNA and amplification by PCR

Digestion using specific restriction nucleases into DNA fragments

separation of DNA fragments by gel electrophoresis

VNTRs are hybridised with DNA probes

the gel is developed, pattern of bands can be seen by placing on an x-ray as probes often contain radioactive tag

this reveals position of bands and banding patterns can be compared between samples

Question 33

What is the process of gel electrophoresis

Answer 33

DNA fragments are placed on a gel plate

the fragments will move towards the positive electrode when switched on as DNA -vely charged due to phosphate groups

smaller fragments move further toward the electrode as are lighter

different sized fragments are separated into band (thicker = more fragments)