Gene Technologies

Question 1

What probe do you use for a recessive disease

Answer 1

Probe complementary to the recessive faulty allele

Need 2

Question 2

What probe do you use for a dominant disease

Answer 2

Probe complementary to the dominant faulty allele

Only need 1

Question 3

Why can protein structure be used to determine evolutionary relationship

Answer 3

Closely related means similar base sequence so similar primary structure of amino acids and therefore protein structure

Question 4

Why is it better to compare DNA base sequence than protein structure

Answer 4

Degeneracy of triplet code

Longer so more specific
DNA base sequence contains introns but protein structure doesn’t

Question 5

Why is it better for restriction endonuclease to be complementary to TATCTGTCTAT than just TAT

Answer 5

TAT repeated too often
Longer one less likely to be complementary to wrong parts
Enzyme is complementary to a lot of DNA for TAT so produces more small fragments

Question 6

Why can plants synthesise insect protein

Answer 6

Genetic code is universal
The same bases exist in all organisms
Same triplet codes for same amino acid
Desired gene can be transcribed into mRNA
And translates into insect protein

Question 7

What does recombinant DNA technology allow

Answer 7

Genes to be manipulated, altered and transcribed from organism to organism
Better understanding of how organisms work
Design new industrial processes and develop medical applications

Question 8

3 important features of the genetic code

Answer 8

Universal: Same triplet codes for same amino acid
Degenerative: More than one base/triplet for each amino acid
Non-Overlapping: Each base only part of one triplet/read once by ribosome

Question 9

5 steps for producing and conducting gene transfer and cloning

Answer 9

Isolation
Insertion
Transformation
Identification
Cloning

Question 10

Benefit of DNA being universal

Answer 10

All organisms have the same triplets coding for same amino acid
So transferred DNA can be transcribed and translates
During protein synthesis

Question 11

3 methods of isolation

Answer 11

Conversion of mRNA to cDNA using reverse transcriptase
Restriction endonucleased to cut a fragment with desired gene
Create gene in gene machine

Question 12

How is reverse transcriptase used in isolation

Answer 12

mRNA mixed with free DNA nucleotides
Enzyme reverse transcriptase added and mixed in
Free DNA nucleotides bind to single stranded mRNA template via complementary base pairing via reverse transcriptase
Into cDNA
Degrade mRNA with RNAase/strong alkali to leave cDNA (single stranded)
Added DNA nucleotides and DNA polymerase to make cDNA double stranded

Question 13

Advantage of using reverse transcriptase in isolation

Answer 13

mRNA in cytoplasm already been spliced so all introns removed
mRNA easier to obtain
Use mRNA base sequence to work out sequence of exon bases without the need for splicing
If being actively expressed, cytoplasm will contain lots of mRNA (e.g insulin mRNA abundant in beta cells of pancreas)
Cells only contain 2 alleles for a gene
Bacteria don’t have enzymes to deal with introns

Question 14

Use of restriction enzymes in isolation

Answer 14

Cut/hydrolyse phosphodiester bonds in DNA
At specific recognition sequences complementary to their active site
These are palindromic sequences (base pair reads same both ways)
If target gene has recognition sequence before and after
DNA incubated with specific restriction endonuclease
Same enzyme must be used for DNA of interest and plasmid so sticky ends match and can form complementary base pairs via H bonds

Question 15

Where do restriction endonucleases cut

Answer 15

Specific recognition sequence complementary to active site
These are palindromic sequences
Read the same in opposite way

Question 16

What two things do restriction endonucleases produce

Answer 16

Sticky ends

Blunt ends

Question 17

What are sticky ends

Answer 17

Transformation

Question 18

Is Sam amazing

Answer 18

Yes

Question 19

What are sticky ends

Answer 19

Overhand and complementary
Same restriction endonucleases
Needed in transformation and modification

Question 20

What are blunt end uses

Answer 20

PCR
Gel electrophoresis
Restriction mapping

Question 21

Regulatory genes

Answer 21

Gene that regulates the expression of one or more structural genes
By controlling the production if a protein
Which regulate their rate of transcription

Question 22

Proteome

Answer 22

Entire set of proteins that is or can be expressed by a genome, cell, tissue or organism at a certain time

Question 23

Gel electrophoresis

Answer 23

Method for separation and analysis or DNA, RNA and protein and their fragments based on their size and charge

Question 24

Oligonucleotide

Answer 24

Small overlapping single strands of nucleotides that are assembled into the desired gene

Question 25

Advantage of gene machine

Answer 25

Easily transcribed and translated by prokaryotes as they have no introns in their DNA

Question 26

Explain how the gene machine works

Answer 26

Desired nucleotide sequence inputted into computer Synthesis of oligonucleotides Which are overlapped And joined together and made double stranded by PCR Gene is inserted into bacterial plasmid and cloned Sequenced, those with errors rejected

Question 27

Vector

Answer 27

DNA carrier (bacterial plasmid/virus) used to transfer foreign DNA into cells

Question 28

How is gene inserted into vector

Answer 28

Use same restriction endonucleoase to isolate DNA fragment and cut open DNA vector So that it has a promoter and a terminator So RNA polymerase can bind before the gene of interest and release after it Producing complementary sticky ends Target DNA fragment anneals to vector DNA by complementary base pairing between the sticky ends DNA ligase uses to join DNA fragment and vector DNA at the sugar phosphate backbone (ligation) Phosphodiester bonds formed New recombinant DNA

Question 29

Two vectors

Answer 29

Plasmid | Bacteriophage/virus

Question 30

Two ways to encourage DNA vector to be transferred into host

Answer 30

Giving bacteria a short electrical shock Opens temporary small pores in the cell membrane Calcium phosphate makes temporary pores in cell membrane

Question 31

Method for in vivo cloning

Answer 31

Cut desired gene from DNA desired organism Using restriction endonuclease Use mRNA from cell of organism Reverse transcriptase from desired DNA ``` Make artifical DNA with correct base sequences Using DNA polymerase Cut plasmid open With same restriction endonuclease Producing stick ends DNA ligase used to join Return plasmid to bacterial cell ```

Question 32

Why must organisms be identified

Answer 32

Not all vectors take up target DNA and become recombinant Not all host cells become transformed by taking up recombinant vectors Only transformed host will synthesis desired protein Only want to clone transformed cells

Question 33

Marker gene

Answer 33

Allows easy identification of cells that have taken up a genetically transformed plasmid Those that have not will not be fluorescent/identifiable

Question 34

3 ways to use marker genes

Answer 34

Antibiotic resistance Fluorescence Enzyme markers

Question 35

How do you identify colonies that have taken up recombinant DNA with replica painting/stamp

Answer 35

Incubate on a growth medium Sterile velvet surface used as a stamp To transfer cells from each colony onto replica plates One has the normal medium but one has the condition which only those transformed can survive in Clone those successful

Question 36

Explain the outcomes for replica painting/stamp

Answer 36

Some cells haven't taken up plasmid So killed by both antibiotics Some cells have taken up original plasmid So resistant to both types Some taken up transformed plasmid So resistant to one but not the other since the gene is cut and disrupted by inserting foreign DNA

Question 37

Primer

Answer 37

Short pieces of single stranded DNA Complementary base sequence to bases at the start of DNA fragment you want to isolate Prevent strands sticking back together Allow DNA polymerase to attach and join free nucleotides together

Question 38

Purpose of PCR

Answer 38

Used to amplify a single DNA fragment into millions of copies Stages are automatically repeated many times Number of DNA molecules doubles every cycle So rapid and efficient

Question 39

PCR

Answer 39

Polymerase Chain Reaction

Question 40

3 steps for PCR

Answer 40

Separation Annealing Synthesis

Question 41

Why is the PCR cooled

Answer 41

Primer won't attach at 95 so H bonds can form

Question 42

Steps for PCR

Answer 42

Heat DNA to 95 degrees which breaks weak hydrogen bonds and separates strands Add primers and nucleotides Cool to 50 degrees to allow binding of nucleotides and primers Add thermostable DNA polymerase (Taq) Heat to 75 degrees DNA polymerase joins nucleotides together by phosphodiester bonds Repeat cycle many times Amplify DNA

Question 43

Number of DNA molecules after each PCR

Answer 43

2^n n=number of cycles

Question 44

Number of cycles in PCR

Answer 44

Log2(n) n=number of DNA molecules

Question 45

Advantage of using a thermostable enzyme in PCR

Answer 45

Enzyme is stable even at high temperatures So not denatured at high temperatures Making it efficient at amplifying DNA

Question 46

Disadvantages of in vivo cloning

Answer 46

Slower - One DNA replication per cell division so limited by growth of cell Cant be used to copy partly broken down DNA Not very sensitive so need a large DNA sample to start with Need to isolate DNA fragment from DNA before it can be inserted into host

Question 47

Advantages of in vivo cloning

Answer 47

Almost no risk of contamination Cells have mechanisms for correcting errors made when copying genes so much more accurate Can be used to clone large DNA fragments Can be used to produce protein or mRNA from the inserted DNA as well as target DNA

Question 48

Disadvantages of in vitro cloning

Answer 48

Can only be used to copy DNA Lacks error-correcting mechanisms so error rate higher Cloning becomes unreliable when used to copy DNA fragments longer than 1000 base pairs

Question 49

Advantages of in vitro cloning

Answer 49

Rapid - can produce millions of copies of DNA in hours Only replicates target DNA so no need to isolate DNA fragment from DNA Can be used to copy partly broken down DNA Very sensitive, only requires a small sample of DNA to start with

Question 50

Three uses/benefits of recombinant DNA tech

Answer 50

Agriculture; GE for high yield, more nutrients, pest resistance, drought and temperature resistant Industry; GE organisms to make large quantities of enzymes quickly and cheaply for industrial processes Medicine; GE organisms can be produced to make large quantities of drugs and vaccines quickly and cheaply

Question 51

Issues of recombinant gene technologies

Answer 51

Antibiotic resistance marker genes within GE could lead to transfer of these genes to pathogens and decrease antibiotic effectiveness Introducing herbicide resistance genes could result in transfer to wild species when they interbreed and produce weeds resistant to herbicides

Question 52

Why are viruses or liposomes used as vectors

Answer 52

Plasmids cannot carry DNA into human cells

Question 53

Liposome

Answer 53

Lipid droplet like a vesicle that carries DNA into cell Fuses with cell surface membrane and releases DNA into cell DNA doesn't move into nucleus so daughter cell won't have functional gene

Question 54

Somatic vs germline gene therapy

Answer 54

Somatic is DNA transfer to normal body tissue Germline is DNA transfer to cells that produce eggs or sperm Somatic gene therapy restricted to patient and isn't inheritable

Question 55

VNTR's

Answer 55

Variable Number Tandem Repeats | Not the same as introns since not in the gene

Question 56

Describe how genetic fingerprinting is carries out

Answer 56

DNA extracted from sample Cut into segments using restriction endonucleases Leaving VNTR's intact DNA fragments separated using gel electrophoresis Mixture put into wells on gel and electric current passed through Immerse gel in alkaline solution/two strands separated Southern blotting DNA fixed to nylon/membrane using UV light Radioactive probe added which is picked up by required fragments Identified using X-ray film/autoradiography