gene tech

Question 1

genome

Answer 1

complete set of genes in a cell. can include:
- genes coding for proteins
- regulatory genes (ie: those coding for TF, siRNA/miRNA)
- mitochondrial dna
- chloroplast dna
- genes coding for antibiotic resistance located in plasmids
- viral RNA/DNA in capsid of virus

Question 2

PHYLOGENETICS

Answer 2

automated sequencing of genomes of species; comparing DNA base sequences and amino acid sequences

Question 3

proteome

Answer 3

full range of proteins a cell can produce

Question 4

recombinant/transgenic dna

Answer 4

cell having two or more sources of dna

Question 5

describe how dna is replicated inside of a cell

Answer 5

dna strands separate
parent strand acts as template
complementary base pairing
dna polymerase joins adjacent nucleotides via phosphodiester bonds
consensation reaction
5’ to 3’ direction
semi conservative replication formed by
each new molecule has 1 template and 1 new strand

Question 6

describe how the polymerase chain reaction (PCR) is used to amplify a dna fragment

Answer 6

taq dna polymerase
add nucloetodies and primers
heat to 95 degrees to break H bonds and seprate strands
reduce temp to 55 degrees so primers bind to dna strand
increase temp to 70 so dna polymerase can add the nucleotides

Question 7

why is the dna heat to 95 degrees during PCR`

Answer 7

produces single strand dna
breaks weak hydrogen bonds between strands

Question 8

why do you add primers during PCR

Answer 8

complementary to end of fragment
replication of base sequence (from there)
prevents strands annealing

Question 9

isolating dna fragments

Answer 9

mrna converted to cdna via reverse transcriptase
use restriction enzymes to cut a fragment
create the gene in a gene machine
dna polymerase makes dsCDNA

Question 10

advantages of reverse transcriptase

Answer 10

mrna easier to obtain
introns have been removed, exons spliced together
bacterial dna doesnt contain introns

Question 11

restriction endonucleases

Answer 11

hydrolyse dna at specific recognition base sequences
- complementary to enzyme active site
- recognition sites are palindromic, base pair read the same in opposite directions

Question 12

what will happen if the recognition sequences for the restriction endonucleases occurs within the dna fragment you want to isolate

Answer 12

will cut this gene and it will not code for a functional protein
cuts are either blunt/sticky end

Question 13

blunt ends

Answer 13

used in pcr and gel electropheresis

Question 14

sticky ends

Answer 14

used in transformation

Question 15

gene machine

Answer 15

• desired nucleotide sequence fed into the computer
• synthesis of short sequences of nucleotides (oligonucleotides)
• loigonucleotides overlapped and made ds via PCR
• ## gene inserted into bacterial plasmid

Question 16

benefits of using gene machine

Answer 16

dna without introns
faster than enzyme controlled reactions
artificial genes easily transcribed and translated by prokaryotes

Question 17

vector

Answer 17

dna carrier (ie: virus, plasmid of bacteia) used to transfer forign dna into cells

Question 18

insertion of genes via vector

Answer 18

isolated target dna fragment inserted into vector dna by cutting open vector dna using SAME restriction endonucleases to produce specifically complementary “sticky ends”
- complementary base pairing of sticky ends between the vector dna and target fragment dna
- dna ligase joins dna fragment and vector dna (ligation via phophodiester bonds)
- new recomnincant dna

Question 19

outline a method for in vivo cloning

Answer 19

vivo = living
- cut desired gene from organism via restriction endonucleases
OR use mRNA from cell via reverse transcriptase to form cDNA
THEN
- make artificial dna with correct sequence of bases
- using dna polymerase
-cut plasmid open
- with same restriction endonucleases
- sticky ends complementary to each other and attach
- dna ligase joins
- return plasmid to bacterial cells

Question 20

marker gene

Answer 20

allows easy identification of cells that have taken up genetically transformed plasmid
common types:
- fluorescence
- antibiotic resistance
- enzyme markers

Question 21

indeitfying transormed bacteria using antibiotic resistance genes

Answer 21

no plasmid = killed by both antibiotics
original plasmid = resistant to both antibiotics
transformed plasmid taken up = resistant to one antibiotic but not second (2nd cut and disrupted by inserting foreign dna)

Question 22

primers

Answer 22

short pieces of ss dna with complemenary base sequences atg start of dna fragment. prevent dna strands sticking to each other.

Question 23

describe the process of pcr

Answer 23

heat dna to 95 degrees breaks weak hydrogen bonds
- add primers and add nucleotides
- cool to 50C to allow binding of primers/nucleotides
- add taq dna polymerase
heat to 75C
- dna polymerase joins nucleotides together
- repeat cycle many times

Question 24

formula for number of dna strands after x number of cycles

Answer 24

2^n where n = number of cycles
no of cycles = log2 (no of dna molecules)

Question 25

why does the exponential curve increase in dna molecules start to plateaue after 35 cycles?

Answer 25

low conc. of nucleotides and primers dna polymerase cant bind to nucleotides SO less new strands of DNA formed as dna polymerase needs primers to start strand synthesis

Question 26

explain why base pairs is a suitable unit for measuring the length of a dna piece

Answer 26

dna = 2 chains AT, CG - bases are a constant distance apart - each base pair is the same length

Question 27

a deletion mutation occurs in gene 1. describe how a deletion mutation alters the structure of a gene.

Answer 27

- removal of one or more nucleotides -frameshift

Question 28

describe the main stages in the copying, cutting and separation of the dna

Answer 28

heat dna to 95C strands separate cool so primers bind to dna add dna taq polymerase restriction enzymes cut dna at specific base sequences use of electric current/gel shorter fragments move further

Question 29

describe a plasmid

Answer 29

circular dna separate fro main bacterial dna contains only a few genes

Question 30

suggest one reason why dna replication stops in the pcr

Answer 30

limited number of primers dna polumerase denatures eventually

Question 31

suggest why the restriction enzyme has cut the hujman dna in many places and the plasmid dna only once

Answer 31

- enzymes only cut at specific base sequences - occurs once in plasmid and many times in dna humans

Question 32

describe how the bacteria containing the insulin gene is used to obtain sufficient insulin for commercial use

Answer 32

- use of fermenters - provides nutrients - named environmental factor - reproduction of bacteria - insulin accumulates and is extracted

Question 33

explain what is meant by a vector

Answer 33

carrier of dna into cell/organism

Question 34

in vivo cloning vs in vitro cloning

Answer 34

vivo: vitro slower, one dna rep per cd: quicker, millions off copies dna/rna/proteins modified, introns removed: no modification corredcting error mechanisms, more accurate: lacks error correcting method

Question 35

benefits of recombinant dna

Answer 35

develop medical applications (spread insulin) - aricultural applications (plants resistant to disease and weather)

Question 36

viruses as vectors

Answer 36

can cause immune response and formation of cytotoxic t cells

Question 37

germ line therapy cons

Answer 37

DNA transfer to cells producing gametes - imperfect solution - denial of human rights, no consent - abuse, favourable characteristics

Question 38

somatic gene therapy cons

Answer 38

dna transfer to normal tissue - not all cells take up recombnant dna - not all cells xpress dna allele - only some tissue types are accessible - multiple treatments - immune response to vector

Question 39

explain how modified plasmids are amde from genetic engineering and hjow the use of the markers enable bacteria containing these plasmids to be detected.

Answer 39

isolate target gene from organism via restriction endonucleases/ rna from otger cell using reverse transcriptiase - get dna to produce sticky ends use dna ligase to join taret dna gene to plasmid also include markwer gene (ie: antibiotic resistance) - add plasmid yo bacteria to grow bacteria expressing marker gene wll have target gene too

Question 40

mrna may be described as a polymer. explain why.

Answer 40

made of of many similar monomers

Question 41

what is a dna probe

Answer 41

short ss of dna bases complementary with dna/allele fragment to be located radioactive/labelled by fluourescence.

Question 42

name 3 techniques used by scientists to compare dna sequences

Answer 42

- pcr - dna fingerprinting -gel electropheresis

Question 43

dna hybridisaton

Answer 43

dna probe binds to complementary target dna - positive= - crrier for recessive allele for genetic disorders - mutation in known genes linked to increase risk of a disease.

Question 44

vntrs

Answer 44

variable number tandem repeats - region of dna between genes - differences in vntrs can be analysed via gel electropheresis

Question 45

describe the process of dna fingerprinting

Answer 45

- dna extracted from sample - dna hydrolysed by restriction endonucleases - produces blunt ends, cut outside of vntrs to leave intact - separate fragments according to size via gel electropheresis - by putting dna in a well and applying current - make fragments ss by putting in strong alkali - add radioactive gene probes to allow positions of fragmenrs to be visualised as bands - put nylon on top fix ss dna to nylon with uv light - identify base sequence of interest using x ray film (autoradiography)