8 genetic technology

Question 1

define recombinant DNA

Answer 1

DNA made by joining pieces from 2 or more different sources

Question 2

write an overview on gene transfer

Answer 2

1- the required gene is identified
it may be cut from a chromosome, made from mRNA by reverse transcription, synthesizd by nucleotides
2- multiple copies of the genes are made by PCR
3- gene is inserted into a vector (plasmid, liposome, viruses)
4- vector inserts the gene into the cells
5- cells with new genes are identified and cloned

Question 3

how can recombinant DNA be made

Answer 3

It is because of the universal genetic code that rDna can be formed

Question 4

what do genetic engineers need

Answer 4

1- enzymes
restriction endonucleases, ligase, reverse transcriptase
2- vectors
plasmids, virus, liposomes
3- markers, genes that code for easily identifiable substances that can be used as markers, GFP ( glows under UV light) or GUS beta glucuronidase enzyme(colorless to colorl)

Question 5

what is the role of restriction endonucleas in bacteria

Answer 5

is to restrict viral infection by recognizing + breaking down the DNA of bacteriophages, it can do this buy cutting the phage DNA into smaller pieces or cutting teh sugar phoshate backbone of DNA . it can give blunt or sticky ends.

Question 6

how is bacterial DNA protected from restrictio endonuclease

Answer 6

by chemical markers or by not having target sites for resriction endonucleas to bind

Question 7

state all the enzymes used and their roles

Answer 7

Restriction Endonucleases-
cuts the DNA strand so that the desired gene can be isolated or inserted into a vector
Reverse transcriptase: reverses transcription to form a single strand complementary DNA cDna FROM mRna coding for the desired gene
DNA polymerase: used to convert the single strand cDNA into double strand dna molecule of the desired gene
DNA ligase- used to insert the gene into the vector
cataylsyes the formation of phosphodiester bonds in the DNA sugar phophate backbone

Question 8

what two enzymes are used in the preparatio of a plasmid vector

Answer 8

restriction endonucleases, DNA ligase

Question 9

how is a plasmid prepared

Answer 9

1- human gene is isolated and treated with R.E whic cuts the DNA and forms stickt ends
2- bacteria is treated with R.E wghich cuts plasmid, creatig sticky ends
3- open plasmids and cut gene are mixed together, complimentary base pairing occurs in which sticky ends form H bonds with complimentary sequences
4- DNA ligase seals gap in sugar phosphat backbone, producing a closed cirle of doble stranded DNA containing the new gene , this is now recombinat DNA

Question 10

how are recombinant plasmids transferred into bacteria

Answer 10

through a process called transformation, recombinant plasmids are mixed with bacteria, bacteria are put into a solution with high CA2+ ion concentraion and are given a heat shock (40 derees), some bacteria take up the palsmid and are said to be trsnformed. this is done to make the membrane mre permeable

Question 11

what do plasmids that produce good vectors have?

Answer 11

circles of DNA- increase stability
small,low molecula mass, redily taken up by bacteria
an origin of replication- can be copied fast
have polylinkeres- short length of dna that have 2 or more sites for restricttion nzymes
have marker genes for screening, allows the cell that has taken up the plasmid to be identified

Question 12

how is insulin produced using genetic engineering

Answer 12

Isolation of human gene:
mRNA that codes for insulin is taken from the pancreas from B-cells in islet of langerhans. this mRNA is incubated w reverse transcriptase. single stranded cDNA formed, using DNA polymerase doube stranded cDNA forms. this cDNA is treated with restriction E.NN preparation of vector:
bacterial plasmid is treated with the SAME restrictin E.N, to create sticky ends
MIXING OF DOUBE STRANDED CDNA WITH STICKY ENDS AND PLASMID WITH STICKY ENDS using DNA LIGASE
to produce recombinant plasmid which is introduced into teh bacteria . transformed bacteria is identified, gene is cloned by growing the bacterium

Question 13

name some advantages of using insulin produced b genetic tech

Answer 13

• identiclal to human inslulin
• more rapid response
• cheaper to produce in large volume
• ethical and religious issues

Question 14

what is a promoter and its uses

Answer 14

is the region of DNA which dtermines which gene will be expressed, it is the site where RNA polymerase binds to in ordeer to begin transcription
the promoter also ensures that RNA polymerase can recognize which is the DNA template strand
- regulates gene expression
-

Question 15

advanatages of flueroscent markers

Answer 15

• easier to identify
• more econmoical = time saving
• • no resistance genes can be passed onto other acteria

Question 16

disadvantages of antibiotic resistan ggenes

Answer 16

• risk of spreading resistan geneto other bacteria
• less effective during trasnformation
• require develeopmont of new antibiotics if no longer effective as markers

Question 17

what is a marker

Answer 17

a marker is a gene that is transfered with the dsesired gene so that scientits can identify whch cells have been succesfully altered or contain recombinant DNa

Question 18

how can antibiotic resistance spread to other bacteria

Answer 18

through transduction (bateria to vrus) or conjugation (bacteria to bacteria)

Question 19

how to use GFP gene to identify trasformed

Answer 19

the GFP gene along w the desired gene are linked with a specific promoter, once promoter activatese adn protein is epressed, transformed bacteria will glow green under UV light

Question 20

name some uses of PCR

Answer 20

since it is used to produce very large quantities of fragments of DNA and RNA from very small quantities, it can be used for DNA profiling to identiyffy criminals, or to determine paternity

Question 21

what is necessary for PCR to occur

Answer 21

• target DNA, primers, DNA polymerase namel TAG polymerase, free nucleotides, buffer solution

Question 22

why is tag polymerase used in PCR

Answer 22

it comes from a thermohphilic bacterium hence it does not denature at high temperatures involved during the firt step of PCR
its optimum temp is high enough to prevent annealing of the DNA STRANDS that have not been copied yet

Question 23

outline the steps ofPCR

Answer 23

1- DENATURATION
2- ANNEALING
3- ELONGATION
1- double stranded dna is heated at 95degrees which breaks the H bonds that bond the two strands together
2- temperatue is decreased to about 65 so that primers can anneal to the ends of the single strands of DNA, by complimentary base pairing, primers redue rebinding of sperated strands
3- temp is incr to 72 which is the optimum temp for TAG polmeraw to create cDNA to produce the new identiacal doube stranded DNA

Question 24

what is gel elctrophoresis

Answer 24

its a technqiue used widely in the analysis of DNA, RNA, protein. during elecrophres, the molecules are seperated due to their net ( overall) charge

Question 25

what causes the seperation in gel electrophoresis

Answer 25

separation occurs because of the:
- of the electrical charge molecules carry, +vely charged molcules move to cathode (negative pole) vice versa. DNA is negatiely charrfed due to phopahte groups and thus when placed in an electric field, moves towards anode
-
different siz molecules move through the gel at different rates. tiny pores in gel cause smaller molcules move faster , while larger molcules move slowly

Question 26

GEL ELECCTROPHREISS PROTEINS DN

Answer 26

mkj

Question 27

use of microarrays

Answer 27

• identift the genes presetn in an organisms genome

- identify which genes r being expressed in cells

Question 28

how is DNA prepared for gel electrophoresis

Answer 28

• DNA is collected, it may be from saliva from a cup, semen, or from a root of the hair.
• multiple copies of the gene are made using PCR
• dna is treated with restriction endonucleases but scientits use enzymes that cut close to VNTRs since VNTRs are part of the non coding part of DNA, and are known to vary between people except for identitcal twins

Question 29

what are VNTRs

Answer 29

Variable number tandem repeats are regions found in the non coding part of DNA that vary between ppl except for indetital twins. they contain variable numbers of repeate DNA sequences.

Question 30

how do we seperate the DNA fragments in gel electrophoresis

Answer 30

1- create an agarose gel plate in a tank. wells are cut into the gel at one end.
2- submerge the gel in an electrolyte solution in the tank
3- insert the DNA fragments into the well using a micropipette
4- generate an electric current. make sure that the cathode is on the side of the DNA fragments as negatviely chargd DNA will move towards anode
5- the smaller mass and shorter pieces of DNA will move faster
6- the fragmnts are not visible and must be transferred to an absorbant paper or nitrocellulose from where they are heated to separate the two strands. probes are added after which an X-ray image is taken or UV light is shown. probes are single straded dna fragments that are complementary to the VNTR regions sought by scientists
7- probes can be identified either by a radioactive label whch causes the probes to emits raditaion that makes the x ray film dark, creating a pattern of dark bands
or a fluorescent tag which glows under UV light

Question 31

whys buffeer solution used in protein seperation

Answer 31

the charge on the R group depends on the pH, hence to keep pH constant

Question 32

state some uses of bioinformatics

Answer 32

once a genome is sequenced, bioinformatics allows scientits to make comparisons with the genomes of other organisms using the many databases online. this can help to the degree of similarity bw organisms

Question 33

what is genetic screening and its uses

Answer 33

it is the testing of the DNA of an embryo, fetus or an adult

Question 34

how have crops been genetically modified

Answer 34

• resistnat to herbicides
  resistant to pests
  enriched in vitamins

Question 35

name benefits of geneting engineering than artifical selection in global demand for food

Answer 35

• organisms w desired charcetrsistiscs are produces more quickly
  no chance that recessive allele will arise in population
  it may come from a different species/kingdom

Question 36

gmo in agriculture

Answer 36

do