Recombinant DNA Tech

Question 1

essential features of recombinant DNA technology

Question 2

give examples of exploitation of recombinant DNA tech

Question 3

cutting DNA

Answer 3

• DNA can be extracted from any organism
• can be cut by restriction endonucleases (enzymes extracted from bacteria)

Question 4

what are restriction endonucleases?

Answer 4

if host bacteria doesn’t want to receive plasmid, they can cut it up using restriction endonucleases

a.k.a. they’re tools bacteria can use to suppress aquisition of plasmids if they don’t want them

Question 5

**how do restriction endonucleases work?

Answer 5

eg. BamH1

= Bacillus amyloliquefaciens enzyme H1

Question 6

joining DNA requires…

Answer 6

• DNA ligase
• this forms phosphodiester bonds
• requires ATP

Question 7

what gives rise to recombinant DNA technology?

Answer 7

the ability to cut & join DNA

Question 8

what can you do with clones in a library?

Answer 8

• can screen for clones containing gene of interest
• various methods e.g. techniques involving DNA/RNA hybridisation
• look for expressed protein with antibody

Question 9

gene library def

Answer 9

collection of recombinant clones

Question 10

power of rDNA tech

Answer 10

• can clone genes from any organism
• can study any individ. gene
• reverse genetics: gene -> phenotype
• In vitro mutagenesis
  -> gene tagging: adding GFP / RFP
  -> reintroduce mutated gene (eg. Drosophila)
• can use gene targeting: In vivo gene disruptions / deletions

Question 11

describe how you could study any individ gene using rDNA tech

Answer 11

• eg. by DNA sequencing -> eg. predicting protein using bioinformatics
• gene expression studies -> techniques involving DNA/RNA hybridisation e.g. microarrays (chips)

Question 12

transgenics

Answer 12

• being able to swap genes between diff sp. has lots of benefits
• because genetics code is universal (all A,T,G,Cs) we can take DNA from 1 organism and put it into another (might need to change codon usage a bit)
• so expression is possible

Question 13

SRY gene

Answer 13

• Sex determining Region Y
• sex-determining gene on Y chromosome in placental mammals (therians)
• this intronless gene encodes transcription factor that is member of SOX (SRY-like box) gene family of DNA-binding proteins
• mutations in this gene give rise to XY females with gonadal dysgenesis (Swyer syndrome).
  -> translocation of part of Y chromosome containing gene to X chromosome causes XX male syndrome

Question 14

TDF

Answer 14

• Therian testis Determining Factor = sex-determining region Y protein / SRY protein …
• which initiates male sex determination

Question 15

useful products produced using rDNA tech

Answer 15

• Insulin
• Factor VIII (essential in blood clotting)
• Tissue plasminogen activator
• Growth hormone
• Vaccine proteins e.g. Hepatitis B
• GM crops: Frankenstein foods?

Question 16

PCR

Answer 16

individ gene amplification without cloning in recombinant E.coli

• In vitro DNA synthesis reaction
• requires target DNA, DNA polymerase & primers
• repeated many times (by heating & cooling) ∴ called ‘chain reaction’
• can amplify 1 gene from a mixture eg. from genomic DNA
• v powerful

Question 17

uses of PCR

Answer 17

• detection of pathogens in water, blood
• genetic fingerprinting
• forensic analysis
• diagnosis of genetic disorders
• prenatal diagnosis
• analysis of ancient DNA
• detection of insecticide resistance (practical 3 on DDT resistance)

Question 18

limitations of PCR

Answer 18

• seq info required to design 2 primers
  -> need to know at least 2 locations quite close together to get info inbetween
• limit on length of amplified fragment (max. ~10Kb fragment)
• significant in vitro mutation rate
  -> polymerase will make mistakes
• v sensitive to exact reaction conditions, so not easily quantified
• tiny amounts of contaminating
  DNA (i.e yours!) will also be amplified