Gene Technology

Question 1

Recombinant DNA

Answer 1

Cell having two or more sources of DNA

Question 2

Similarly between transcription and translation

Answer 2

Both process are universal

Question 3

5 steps of recombinant DNA technology IITIG

Answer 3

Isolation
Insertion
Transformation
Identification
Growth

Question 4

3 ways to isolate a gene

Answer 4

1. Creating oligonucleotides using a known sequence of DNA using a gene machine which overlap each other
2. Using reverse transcriptase which coverts MRNA to cDNA
   3, using restriction endonuclease to cut fragment containing desired gene

Question 5

Gene machine

Answer 5

1. Desired nucleotide sequence is fed into a computer
2. Synthesis if oligonucleotides whaivh a shirt sequences of nucleotides
3. Oligonucleotides overlap each other and join together and made double stranded using PCR
4. Gene inserted into plasmid of bacteria

Question 6

Using reverse transcriptase

Answer 6

Makes cDNA copies from MRNA template
Free DNA nucleotides bind by complementary base pairing by hydrogen bonding on MRNA template
Reverse transcriptase joins DNA nucleotides via condensation reaction forming a phosphodiester bond forming single cDNA molecule. DNA polymerase required to make it double stranded

Question 7

Using restriction endonucleases

Answer 7

Hydrolyses DNA at specific base sequence and is complementary forming sticky ends. They are palindromic

Question 8

Significance of promotion region and terminator region in relation to isolated gene of interest

Answer 8

Add promoter region that will allow transcription factors to bind then allows the gene to be expressed once inserted into plasmid
Terminator region also added a fragment which causes transcription of gene of interest stop

Question 9

Advantage of using gene machine than enzyme catalyst reactions

Answer 9

Faster than all enzyme catalysed reactions

Question 10

Advantage of using reverse transcriptase/Gene machine over restriction endonuclease

Answer 10

Human gene contains intron whereas bacteria cannot remove introns by splicing. Reverse transcripts/ Gene machine produces gene without an introns

Question 11

Advantage of using reverse transcriptase

Answer 11

1. mRNA is much easier to obtain as cell producing large amount of protein will have many mRNA.
2. Mature mRNA can be reversed transcribed into DNA so fragment doesn’t contain introns

Question 12

Vector

Answer 12

DNA carrier used to transfer foreign DNA into cells

Question 13

Insertion

Answer 13

1. Cut open plasmid and gene of interest using same restriction endonuclease so sticky ends are complementary to each other
2. DNA of interest and vector DNA anneals by complementary base pairing by hydrogen bonding
3. Mix with DNA ligase used to join DNA fragment and vector DNA sticky ends parts forming Phosphodiester bonds

Question 14

Why do scientists aim to insert a vector into the cytoplasm of a bacteria cell and the nuclear DNA of human?

Answer 14

1. Bacteria cells divide by binary fission in the cytoplasm as that’s where plasmids are found. Replication of plasmids Replication of circular DNA. Produces genetically identical cells
2. Human cells divide by mitosis which occurs in the nuclear DNA to produce genetically identical daughter cells

Question 15

When inserting a vector into eukaryotes, why do scientists injected into gametes are not somatic body cells?

Answer 15

Zygote cell is Totipotent so divide by mitosis into all type of cell
Therefore gene will be transcribed and translated into proteins

Question 16

Transformation

Answer 16

In bacterial cell they divide by binary fission.
In human cells divide in nuclear DNA by mitosis in early stages so passed onto daughter cells

Question 17

Why use Identification?

Answer 17

1. Not all vectors take up gene of interest to become recombinant
2. Not all host cell become reformed by taking up taking up recombinant DNA

Question 18

Marker gene

Answer 18

Allows easy identification of cells that have taken up transformed plasmid as cells that have taken up gene will glow under UV light

Question 19

In vitro meaning

Answer 19

In a glass

Question 20

What is PCR used for?

Answer 20

To make millions of copies of single fragment of DNA. Number of DNA molecules double with every cycle
2n to work out number of DNA molecules

Question 21

Describe PCR?

Answer 21

1. Heat to 95 degrees to break hydrogen bonds and separate both strands of DNA
2. Add primers which are short single stranded DNA sequences complementary to start of allele
3. Add free DNA nucleotides
4. Cool temperature to 50 degrees to allow binding of printers and nucleotides by complementary base pairing by hydrogen bonds
5. Increase temperature to 80 degrees to allow heat stable taq DNA polymerase to join adjacent DNA nucleotides by condensation reaction forming phosphodiester bonds to produce complementary DNA
6. Realest cycle many times
   2.

Question 22

What is a primer?

Answer 22

Short single stranded DNA base sequence complementary to start of allele

Question 23

Graph for PCR

Answer 23

Exponential increase as DNA doubles every cycle but eventually plateaus as primers / DNA nucleotides have run out

Question 24

DNA probe

Answer 24

Short single strand of DNA that has a base sequence complementary to known base sequence of DNA

Question 25

Why do you make DNA probes radioactive or dyed?

Answer 25

DNA is invisible for radioactive nucleotide gives out light to detect location of gene on chromosomes

Question 26

How do you screen DNA for multiple sequences of gene of interest?

Answer 26

1. Amplification= use PCR to replicate many copies of DNA. 2. Cut DNA using restriction endonuclease to form blunt ends 3. Separate DNA fragments using gel electrophoresis 4. Add DNA probe that binds by DNA hybridisation. 5. Shine UV light to identify DNA of interest or compare position of bands to known DNA sample

Question 27

Describe genetic fingerprinting can be used on a sample of DNA and why can identify a person?

Answer 27

1. Extract DNA from sample and PCR to amplify and cut using restriction endonuclease to make blunt end fragments. 2. Cut must leave VNTR intact 3. Seperate DNA fragments according to length and charge using gel electrophoresis 4. By placing onto well and passing an electric current through 5. Using an alkaline solution to make DNA single stranded. 6. Use of southern blotting to transfer onto nylon membrane. 7. DNA hybridisation by adding radioactiveDNA probes 8. Use X- day film to see 9. Pattern unique to individual

Question 28

Name and explain factors that may slow down the process of obtaining a genetic fingerprint

Answer 28

1. If small amount of DNA.= needs PCR for amplification 2. If DNA sample is contaminated with other DNA.