Recombinant DNA technology

Question 1

How can plasmids be genetically modified and inserted into bacteria

Answer 1

• isolate the wanted gene from the DNA
• using restriction endonuclease to leave complementary sticky ends on the selected gene and the plasmid
• insert the gene into plasmid using ligase enzyme with the addition of a marker e.g. antibiotic resistance
• put the plasmid containing the recombinant DNA back into the bacteria by heating then cooling then heating
• grow on a medium where marker would be expressed e.g. containing antibiotics
• bacteria that grows will have be antibiotic resistant and contain the desired gene

Question 2

Name the type of enzyme used to cut open the plasmid

Answer 2

restriction endonuclease

Question 3

Name the type of enzyme used to insert the gene onto the plasmid

Answer 3

ligase

Question 4

Describe the process of genetic fingerprinting

Answer 4

• DNA is cut into fragments using a restriction enzyme
• DNA fragments are separated according to size by gel electrophoresis under electrical influence
• gel is then immersed into alkali to separate the double strands into single strands (southern blotting) and then transferred onto a nylon membrane
• radioactive/fluorescent DNA probs are used to bind to the complementary base sequence of the VNTRs under specific temperature and pH conditions
• nylon film can be x-rayed and the probs will be exposed, fluorescent probe can be located visually

Question 5

What is the process of PCR (Polymerase Chain Reaction)

Answer 5

increases the amount of DMA in a sample by:

• DNA strand is separated and the DNA fragments, primers and DNA polymerase are placed in a vessel, and heated to 95°C
• the mixture is cooled to 55°C, causing the primers to anneal (join)to their complementary bases at the ends of the DNA fragment
• the synthesis of DNA happens when the temperature is increased to 72°C which is the optimal temperature for the DNA polymerase to add complementary nucleotides to the DNA strands

Question 6

What is a primer of the PCR

Answer 6

• short sequences of nucleotides that have a set of bases complementary to those at one end of the two DNA fragments
• provide a starting point for the DNA polymerase to begin copying the DNA
• prevent two separate DNA strands from rejoining

Question 7

What is in vitro cloning

Answer 7

using method of PCR that is rapid and does not require living cells

Question 8

What is in vivo cloning

Answer 8

isolating the gene of interest and use of gene machine

Question 9

What is meant by the term Variable Number Tandem Repeat (VNTR)

Answer 9

highly repetitive sequences of DNA bases

Question 10

How does the structure of a genome allow it to be used for genetic fingerprinting

Answer 10

• VNTRs differ in size/length between different individuals
• VNTRs occur in lots of different places in the genome between different individuals
• the difference in size and location of VNTRs creates a genetic fingerprint unique to the individual which can be used to identify them

Question 11

What is southern blotting used for

Answer 11

to separate double-stranded DNA, to make it single-stranded for the DNA probe to bind

Question 12

How is a gene made using a gene machine

Answer 12

• the amino acid sequence os determined
• the nucleotide triplets are worked out and the base sequence is fed into a computer
• the computer designs gly

Question 13

Describe the following stem cells:
Totipotent
Pluripotent
Multipotent
Unipotent

Answer 13

totipotent - can differentiate into any type of cell, found in early embryo

pluripotent - can differentiate into almost any type of cell, found in embryonic and fetal stem cells

multipotent - can differentiate into limited number of specialised cells, found in adult and umbilical cord blood stem cells

unipotent - can differentiate into a single type of cell, found in adult tissue

Question 14

What are induced pluripotent stem cells (iPS cells)

Answer 14

• they are very similar to embryonic stem cells (pluripotent cells) however they can potentially divide indefinitely
• can overcome ethical issues surrounding use of embryonic stem cells
• can be used to regrow tissues

Question 15

What is decreased acetylation

Answer 15

• acetyl group is removed from molecule
• increases positive charges on histones and therefore increases attraction to the phosphate groups of DNA
• DNA becomes less accessible to transcription factors to initiate mRNA productions

Question 16

What is increased methylation

Answer 16

• methyl group is added to molecule
• inhibits transcription by making DNA inaccessible to transcription factors
• by attracting proteins that condense the DNA-histone complexes

Question 17

What are causes of uncontrollable cell devision

Answer 17

proto-onco genes that mutate are called oncogenes:

• this oncogene may code for a receptor that does not need a growth factor
• or oncogene may code for to much growth factor

a mutated tumour suppressor gene may cause cell devision not to be switched off thus not coding for an inhibitor of a growth factor

Question 18

Why are they a variety of different primers used in an experiment

Answer 18

as the base sequence of nucleotides differ, different complementary primers are needed

Question 19

Why would DNA replication in the PCR stop

Answer 19

as the primers/nucleotides run out