8.4 - Gene technologies

Question 1

What is recombinant gene technology

Answer 1

transfer of DNA fragment from one organism to another

Question 2

3 reasons why recombinant DNA technology works

Answer 2

1. genetic code is universal so same codons specify the same amino acids in all organisms
2. mechanisms for transcription and translation are universal genes of one species can be expressed in another
3. inserted gene can be transcribed into mRNA and translated into a functional protein in host cell

Question 3

summarise process of reverse transcriptase to produce DNA fragments

Answer 3

mRNA complementary to target gene is used as template

mixed with free nucleotides which match up base pairs

reverse transcriptase forms the sugar phosphate backbone

cDNA is produced

DNA polymerase makes cDNA double stranded

• cDNA has no introns as it is mRNA template

Question 4

summarize use of restrictions endonucleases in production of DNA fragments

Answer 4

• restriction endonucleases cut DNA at specific sequence at RECOGNITION SITE

-different RE cut at different points

• blunt or sticky ends are formed

Question 5

two ways we can amplify DNA fragments (bullets)

Answer 5

. In vitro with PCR
.In Vivo with host cells, vectors, plasmids ect…

Question 6

what is the reaction mixture in the first stage of PCR

Answer 6

mixture consists of DNA fragments(to be amplified), primers ( complementary to start of fragment) , free nucleotides (to match exposed bases) , DNA polymerase (to create new DNA strand)

Question 7

summarise PCR DNA fragment amplification

Answer 7

• solution consisting of sample DNA, primers, free nucleotides, DNA polymerase

-solution is heated to break hydrogen bonds separating DNA strands

• ANNEALING temperature is cooled to 50-60c so primers can bind to strands

-temperature increased again to 70c so taq polymerase can pair bases to free nucleotides

-process repeated until enough DNA is made

Question 8

summarise in vivo DNA fragment amplification

Answer 8

• plasmid is used as a vector
  -cut with the same restriction endonucleases as DNA
  -so ends are complementary
  -DNA ligase joins fragment with plasmid

RECOMBNINANT DNA is formed

Question 9

how are vectors inserted into host cells

Answer 9

-cell transformation
-host cells mixed with vectors in ice cold solution of calcium chloride to make walls more permeable
-then heat shock
-encourages cells to take up vectors
-cells can be grown and DNA fragments cloned

Question 10

summarise how to identify transformed cells

Answer 10

marker genes (coding for fluorescence/antibiotic resistance/certain enzymes ect) are also inserted into vectors
UV light or antibiotics can be used to identify what has taken up the light

Question 11

how can DNA probes be used to locate specific alleles

Answer 11

probe is designed so sequence is complementary to allele you want to find

labelled, amplified using PCR, then added to a sample of single stranded DNA

probe will bind if allele present

Question 12

3 reasons for genetic probing

Answer 12

to screen someone’s DNA for health conditions

to identify a gene for genetic engineering use

to predict how someone will respond to a drug

Question 13

what is the purpose of hybridisation

Answer 13

to measure degree of difference between two strands of DNA

can be used to compare someone’s DNA to a certain gene to see if they have it

Question 14

summarise the process of DNA hybridisation

Answer 14

-one DNA strand is labelled and mixed with an unlabelled comparison strand

-the more similar the strands the more strongly they bind and more energy required to break strands apart

Question 15

benefits of genetic profiling

Answer 15

• can identify heritable diseases very early and begin treatment

-reduce impact on individuals

-make treatments more effective

Question 16

what is genetic fingerprinting

Answer 16

teqnique used to compare two DNA samples

Question 17

brief summary of genetic fingerprinting

Answer 17

DNA sample obtained
VNTRs curt out using restriction endonucleases

labelled and cloned using PCR

Fragments separated using gel electrophoresis

banning patterns of each sample can then be compared

Question 18

explain gel electrophoresis

Answer 18

DNA fragments are placed at one end of gel in a buffer solution

current is passed through DNA sample and moves to cathode

shorter fragments travel further and faster
pattern of bands created is unique to individuals

Question 19

what is a DNA probe

Answer 19

a short single stranded piece of DNA complementary to a specific allele or gene

Question 20

what technique is used to increase the likelihood of bacterial cells take up the desired gene

Answer 20

electroporation

Question 21

what are microorganisims that have taken up the recombinant plasmid

Answer 21

transgenic

Question 22

what are the names of the 3 steps involved in PCR and the temperatures at these times

Answer 22

Denaturation: 95c

Annealing: 55c

Elongation: 72c

Question 23

how are DNA fragments cloned before genetic in genetic fingerprinting

Answer 23

PCR machine

Question 24

what are the uses of genetic fingerprinting

Answer 24

• determining genetic relationships
• forensic science
• animal and plant breeding
• medical diagnosis

Question 25

how can DNA fingerprinting be used in forensic science

Answer 25

suspect DNA fingerprinting matches DNA sample at crime scene

Question 26

how can DNA fingerprinting be used in medical diagnosis

Answer 26

- detect inherited genetic disorders by comparing parent DNA to embryonic stem cells - tumour diagnosis

Question 27

how can genetic fingerprinting be used in animal and plant breeding

Answer 27

detect how closely related two individuals of the same species are to prevent interbreeding