6.3 Manipulating Genomes

Question 1

what does dna polymerase do

Answer 1

Adds complementary nucleotides to form new DNA strands
Works 5′ → 3′ only
Corrects mistakes to maintain accuracy
Continuous on leading, discontinuous on lagging

Question 2

describe Fred Sanger’s DNA sequencing approach

Answer 2

use single strand of DNA as template for 4 different experiments in separate dishes and each dish contained a solution with the 4 different bases and dna polymerase
to each dish , a modified version of one of the DNA bases was asses (modified = once added in the complementary strand, no further bases could be added) and each base had a radioactive isotope

Question 3

what happened as Sanger’s reaction progressed

Answer 3

1000s of dna fragments of varying length were generated and they were passes through a gel by electrophoresis
Smaller fragments travelled further and so they were alls sorted by varying length
Nucleotide bases at the end of each fragment was read according to its radioactive isotope

Question 4

how does chain termination work

Answer 4

single strands of DNA are broken into chunks of every possible length and the final base is tagged with a radioactive label
the radioactive tag at the end of each length of dna is read and the pieces are lined up in order of length to generate the sequence

Question 5

the gene that needed to be sequenced, how was is isolated

Answer 5

using restriction enzymes from a bacterium

Question 6

how was dna cloned in Sanger’s process

Answer 6

dna inserted into bacterial plasmid and then into E coli bacterium host and then cultured and then DNA is coped many times
each new bacterium contained a copy of the candidate gene and then the lengths of DNA were isolated using plasmid preparation techniques and then sequenced

Question 7

overall, what is the name of the technique Sanger used to sequence DNA

Answer 7

chain termination

Question 8

what type of method does pyrosequencing use

Answer 8

sequencing by synthesis

Question 9

overall summary of pyrosequencing

Answer 9

synthesising a large strand of DNA complementary to the strand to be sequenced, one base at a time, whilst detecting by light emissions, which base was added at each step

Question 10

what is a nucleoside

Answer 10

A nucleoside is a molecule made of a nitrogenous base attached to a pentose sugar, but without any phosphate group

Question 11

despite humans and other organisms having very similar genes, why are there still differences in terms of genetics

Answer 11

some shared genes have been subtly changes through changes to the regulatory dna regions
regulatory and coding regions interact so they do not increase the number of genes but can increase the number of proteins

Question 12

all humans are genetically similar, however there are different alleles and around 0.1% of DNA is not shared with other - how?

Answer 12

DNA sequences can differ due to random mutations like substitutions

Question 13

where on the dna can these random mutations occur

Answer 13

single nucleotide polymorphisms or SNPs
some have not effect on the protein and some can alter a protein or alter the way ran regulates the expressions of another gene

Question 14

examples of synthetic biology

Answer 14

information storage, medicine production, novel proteins, biosensors, nanotechnology

Question 15

what are tandem repeats

Answer 15

repetitive segments of dna that do not code for proteins and each tandem has the same core sequence just with an alternative 1 nucleotide

Question 16

what did Jeffreys realise from his tandem analysis

Answer 16

than number of tandem repeats showed family resemblance but the DNA profile for each family member unique

Question 17

what is the procedure of dna profiling

Answer 17

1. dna is obtained from an individual (through saliva, mouth swab, blood or hair)
2. dna digested with restriction enzymes that cut dna at specific recognition sites and then cut into fragments which vary in size from individual to individual
3. fragments are separated by gel electrophoresis and stained and larger fragments travel the shorted distance in the gel
4. banding pattern can be seen and dna is compared with other individuals in the same processing as above

Question 18

what type of dna is used in dna profiling

Answer 18

short tandem repeats sequences (STR) of DNA
the exact number varies from person to person

Question 19

where can dna profiling be applied

Answer 19

forensic science - identify criminals, identify remains, victim’s body parts after crashes or other disasters, match profiles from descendants of those lost during WW1 on battlefield remains, maternity and paternity tests, analysis of disease from protein electrophoresis in type of haemoglobin for sickle cell anaemia or Huntington’s

Question 20

what is polymerase chain reaction (PCR)

Answer 20

biomedical technology in molecular biology that can amplify a short length of dna to thousands of million of copies

Question 21

what are the 4 principles PCR relies on

Answer 21

1. DNA is made of 2 antiparallel backbone strands
2. each strand of DNA has 5’ end and a 3’ end
3. DNA only grows from the 3’ end
4. base pais pair up accordingly to complementary base pairing rules (at cg)

Question 22

how does PCR differ form DNA replication

Answer 22

1 only short sequences of up to 10000 base Pais can be replicated - not entire chromosomes
2. it requires the addition of primer molecules to make the process start
3. a cycle of heating and cooling is needed to separate the dna strands, bind primers to the strands and for the dna strands to be replicated

Question 23

what enzyme is used in PCR

Answer 23

Taq DNA polymerase

Question 24

what are the steps for PCR

Answer 24

1. sample of DNA mixed with DNA nucleotides, primers, mg2+ ions and enzyme Taq DNA polymerase
2. pictures heated to around 94-96 degrees to break h bonds between complementary. base pais and therefore denature DNA double strand into 2 ingle dna strands
3. mixtures is cooled so the primers can anneal (bind by hydrogen bonding) to one end of each single dna strand (gives small section of double stranded dna molecule at the end of each singular strand)
4. Tag DNA polymerase enzyme bonds to the ends of the trend where it is double stranded (optimum temp is 72)
5. temp is raised to 72 degrees which keeps the dna strand single
6. Taq dna polymerase catalyses the addition of dna nucleotides to single stranded dna molecules starting at the end of the primer in the 5’ to 3’ direction
7. once Taq dna polymerase reaches the other end of the dna molecule then a new double strand of dna has been generated
8. the whole process begins again and is repeated for many cycles

Question 25

mathematically, how does the amount of dna increase

Answer 25

exponentially: 1 - 2 - 4 - 8 - 16 - 32 - 64 - 128

Question 26

where is PCR applied

Answer 26

tissue typing - donor and recipient tissue can e typed to reduce refection risk of transplant detection of oncogenes - mutation identified and meds can be tailored in that approach detection mutations identifying viral infections monitoring spread of infection disease forensic science - dna profiling research purposes