Manipulation of DNA, Use in Therapy and Diagnostics

Question 1

Definition of recombinant DNA

Answer 1

Contains DNA from more than 1 organism

Question 2

Definition of restriction endonuclease

Answer 2

cut DNA by recognizing specific nucleotide sequences and cuts both strands in sugar phosphate backbone

Question 3

Definition of cDNA

Answer 3

DNA copy of mRNA produced by reverse transcriptase that contains no introns

Question 4

Definition of PCR

Answer 4

Alternative method to make many copies of specific DNA fragments without vectors

Question 5

Definition of microarray

Answer 5

Use nucleic acid hybridization to rapidly ,ensure which genes are expressed in tissue sample

Question 6

Definition of ssDNA/oligonucleotide

Answer 6

Section of DNA that matches all the genes in the genome

Question 7

Why is it useful to isolate and manipulate DNA

Answer 7

Isolate, modify genes to understand function
Go from mutant phenotype => sequence
Manufacture proteins and vaccines
Create transgenic plants/organisms
Diagnose genetic diseases, develop gene therapy, personalized medicine

Question 8

What is recombinant DNA technology

Answer 8

Allows isolation, manipulation of DNA

Gene cloning, copies made of unique DNA strands

Question 9

What are restriction enzymes

Answer 9

Recognize specific nucleotide sequences in DNA, cuts both strands in sugar phosphate backbone
Used in bacteria, protect themselves fro viral DNA
Cut at palindromic sequences to form sticky/blunt ends

Question 10

How is agarose gel electrophoresis used

Answer 10

DNA visualized in gel by adding ethidium bromide, binds to DNA, fluoresces when exposed to UV

Question 11

How would you produce recombinant DNA in bacteria

Answer 11

Large amounts of individual DNA fragments needed
Use bacteria/yeast to replicate, amplify individual fragments
Use plasmids as vector, carry fragments into host

Question 12

How to clone DNA molecules in vivo

Answer 12

Plasmids have replication origin, replicate independently of bacterial chromosome
High no of plasmids maintained in each bacterium, have antibiotic resistance genes
Fragment and plasmid cut with same restriction endonuclease, have same complementary cohesive ends
Cut DNA fragments and cut plasmid mixed and anneal to sticky ends
DNA ligase ligates DNA fragment and plasmid, form recombinant DNA

Question 13

How to amplify DNA in vivo

Answer 13

Individual recombinant plasmids taken up by Ecoli
Using origin of replication, 100-200 caries of recombinant plasmid generated in each bacteria
As bacteria divide, each one contains 100-200 copies of recombinant plasmid

Question 14

Uses of cloned DNA

Answer 14

Allow mapping and sequencing of genes within genome
Identify changes in genome, associated w phenotype, pathology
Characterise organisation, location of repetitive DNA in genome
Genetic engineering
Gene therapy
Express large amounts of protein

Question 15

Problems with cloning eukaryotic genes

Answer 15

Cannot use genomic DNA, has introns
Need to use mRNA, no introns, convert back to DNA (reverse transcriptase)
Harvesting mRNA shows up what genes are expressed in cells

Question 16

Formation of cDNA

Answer 16

Reverse transcriptase forms cDNA from mRNA
cDNA libraries have complementary DNA copies of mRNA present in cell population
cDNA used as introns removed, clone size is smaller

Question 17

Genomic DNA vs cDNA

Answer 17

All sequences, genic and intergenic represented almost equally

Frequencies of genes in cDNA reflect levels of gene expression and include only sequences found in mRNA

Question 18

How to sequence DNA

Answer 18

Use DNA polymerase to copy single stranded DNA
Dideoxynucleotide chain-termination sequencing method used
Dideoxynucleotides interrupt DNA polymerase

Question 19

How is deoxynucleotides different from dideoxynucleotides

Answer 19

3’ is H instead of OH

DNA polymerase cannot act on H, cannot form PDB

Question 20

How to sequence with dideoxynucleotide sequencing

Answer 20

DNA replicated in presence of dCTP and some ddCTP and DNA polymerase
Some fragments end prematurely, length depends on where the ddCTP is incooporated

Question 21

How to use automatic DNA sequencing

Answer 21

All 4 ddNTPs marked with fluorescent markers and dNTPs used
Identify which ddNTP added at end of each fragment by colour, separated in capillary gel
Laser excites fluorescent tag on each fragment, wavelength read, info recorded
Fluorescence pattern produced shows DNA sequence

Question 22

Describe Next Generation Sequencing

Answer 22

Uses sequential addition of nucleotides on microchips

Short fragments, v rapid

Question 23

Use of sequence info

Answer 23

Sequence info for genes in genome
Identify genetic differences between organisms, identify basis of genetic disease
Genomic medicine (100000 Genome project)

Question 24

What is PCR

Answer 24

Amplify DNA fragments without cloning vector, in thermocycler

Question 25

PCR steps

Answer 25

Denaturation
Primer annealing
Primer extension

Question 26

What happens in denaturation

Answer 26

95C
Separate DNA into single strands,
30s

Question 27

What happens in primer annealing

Answer 27

45-68C
Primers hybridize complementary sequences in target DNA
30s

Question 28

What happens in primer extension

Answer 28

72C
Taq polymerase to synthesize DNA
1min

Question 29

Separation of PCR products

Answer 29

Gel electrophoresis

PCR product size = amount of DNA between primers

Question 30

Uses and applications of PCR

Answer 30

Amplify DNA from limited sources
Amplify whole genes, specific DNA regions
Mutation screening for genetic disorders
Forensic, paternity testing

Question 31

How to use DNA microarrays/gene chips

Answer 31

Use nucleic acid hybridization, rapidly measure which genes are expressed in tissue sample
Occurs when complementary DNA strands anneal
All mRNA from sample converted to cDNA, fluorescnetly labelled, denatured into single strands, probe
Probe applied to array which contain spots where single stranded DNA from each gene in genome is laid out
Location of fluorescence= gene expressed in sample
Brightness of fluorescence=level of expression

Question 32

How to use comparative microarrays

Answer 32

Directly compare gene expression in 2 samples w different color probes