Gene Technology

Question 1

Genome

Answer 1

Complete set of genes in a cell

Question 2

Recombinant DNA

Answer 2

A cell having two or more sources of DNA
Possible as genetic code is universal,degenerate and non-overlapping

Question 3

5 steps in recombinant DNA technology

Answer 3

Isolation of genes, insertion, transformation, identification and growth

Question 4

1- isolating a copy of specific DNA fragment
What are the different methods

Answer 4

Gene machine
Reverse transcription
restriction endonucleases

Question 5

1-using reverse transcriptase

Answer 5

Isolated mRNA from donor cell
Add dna nucleotides and reverse transcriptase
Free dna nucleotides bind to single stranded mRNA via complimentary base pairing
Reverse transcriptase joins dna nucleotides together to form single standard cDNA molecule
Add dna polymerase to make cDNA double stranded

Question 6

Advantages of using reverse transcriptase

Answer 6

mRNA is easy to obtain
Using mRNA means introns removed whereas genes contain both introns and exons

Question 7

2-using restriction endonuclease

Answer 7

They hydrolyse dna at specific base sequence (either side of gene)
Regcognition sequence is usually palindromic -based pair is same whether read forwards or backwards
Cut made is blunt(used in pcr and gel electrophoresis)
Or sticky(used in transformation)

Question 8

C-gene machine

Answer 8

Enter the desired sequence of nucleotides into a computer
Creates small sections of overlapping single strands of nucleotides
Joined to make dna
Pcr used to amplify and make double stranded

Question 9

Advantages of the gene machine

Answer 9

Artificial genes are easily transcribed and translated by prokaryotes as they have no introns in their dna

Question 10

What is a vector

Answer 10

DNA carrier

Question 11

Insertion of genes into a vector

Answer 11

cut vector using the same restriction enzyme that was used to isolated dna fragments
-Produces complimentary sticky ends between ends of dna fragment and cut ends of the vector dna
-Target dna fragment anneals to vector dna by complimentary base pairs between their sticky ends
-DNA ligase used to join them at sugar phosphate backbone and forms phosphodiester bonds

Question 12

Transformation

Answer 12

Heat shock/ electric current used to make temporary holes in cell curvaceous membrane
Bacterium takes up vector plasmid
Recombinant organism

Question 13

Identifying transformed host cells

Answer 13

not all vectors take up target dna to become recombinant
Marker genes are added
Fluorescence - fluoresces when exposed to uv light
Enzyme markers - no colour change indicted required gene has been inserted

Question 14

Identifying transformed bacteria using antibiotic resistance genes

Answer 14

Cells that didn’t take up plasmid are killed by both types of antibiotics
Cells that took up original plasmid are resistant to both types of antibiotics
Cells that took up the transformed plasmid are resistant to one antibiotic

Question 15

Cloning in vitro via pcr

Answer 15

Pcr used to amplify
Several stages that automatically repeat
1 seperation - strands separate at 95 c
2 annealing -addition of primers ,primers bond 50c
3 synthesis - makes 2 dna fragments 1old 1 new

Question 16

Primer

Answer 16

Short pieces of single stranded dna with complimentary base sequences to dna fragments

Question 17

Benefits of gene tech

Answer 17

Develop medical applications
Better understanding of biological processes

Question 18

Concerns with gene tech

Answer 18

Introducing herbicide resistant genes to crops
could result in transfer to wild plants
Which could become resistant to herbicides

Question 19

Somatic and germ line gene therapy

Answer 19

Somatic - dna transfer to our normal body tissue
Germ line-dna transfer to cells that produce eggs or sperm

Question 20

Limitations of somatic and germ line gene therapy

Answer 20

Not all cells take up new dna
Multiple treatments may be needed

Question 21

Objections of gene therapy

Answer 21

Tech is imperfect
Denial of human rights
Potential abuse

Question 22

Genetic screening using dna probes and dna hybridisation

Answer 22

-determine nucleotide sequence of the mutant allele that you’re trying to find
Create comp dna probe and add a marker
-many dna probes made using pcr
-dna with suspected alle is heated to separate both strands
-seperated strands are cooled in a mixture of dna probes
If strand has mutant allele probe will bind to it as bases are complimentary
-dna is washed to remove unattached probe
-remaining hybridised dna will be fluorescently binded
-dye is detected by shining light causing fragment to fluoresce

Question 23

Gene probe

Answer 23

Short, single stranded dna molecule with complimentary base sequence to dna fragment

Question 24

Genetic fingerprinting and VNTR’s

Answer 24

Uses the region of dna between genes called VNTR’s
Each person has 2 copies of each VNTR on each homologous chromosome
1 from each parent
Difference in length in VNTR can be identified by gel electrophoresis

Question 25

DNA fingerprinting

Answer 25

-extract dna from the nucleus of cells
-pcr amplifies VNTR regions
restriction enzymes are used to cut dna into fragments
-must make blunt ends
-separate dna fragments using gel electrophoresis
-put dna in well and apply electrical current
-make fragments single stranded by soaking in alkali solution
-add radioactive gene probes with comp base sequences to VNTR
-identify base sequence using x ray film

Question 26

Using liposomes as vectors

Answer 26

Using liposomes as vectors
Lipid droplets which can cross phospholipid bilayer and releases target dna into the cell
However dna does not move into the nucleus so new daughter cells will not have the functional gene