6.3 MANIPULATING GENOMES

Question 1

Define genome

Answer 1

• The complete set of genes in a cell

Question 2

Define sequencing

Answer 2

• Identifying the locus and the order of DNA bases for particular genes

Question 3

Describe what genome sequencing gives information about

Answer 3

• The location of genes
• Evidence for evolutionary links between organisms

Question 4

Define locus

Answer 4

• The location of a gene on a region of chromosome

Question 5

State the three types of sequencing

Answer 5

1) Sanger sequencing
2) Automated sequencing
3) High throughput sequencing

Question 6

Compare the three types of sequencing

Answer 6

• Sanger sequencing is slow but high accuracy and requires manual reading of results
• Automated sequencing uses sanger techniques so slow but accurate however computer reads results
• High throughput allows for whole genome sequencing and is very fast but less accurate and computer reads results

Question 7

Explain why during sequencing:
-Copies of a gene are broken into random fragments
- Fragments are sequenced separately
- Sequences are put back together in correct order by matching overlapping fragments

Answer 7

• Because whole genes and genomes are too large to be sequenced in one fragment

Question 8

State three uses of genome sequencing

Answer 8

• Identify phylogenetic relationships
• Predict primary structure of proteins from genes by predicting the sequence of amino acids in polypeptides
• Create gene probes for disease screening in synthetic biology

Question 9

State what gene probes are

Answer 9

• Radioactive/fluorescent fragments of DNA that are complementary to known target sequences of DNA
• (Are easily identifiable)

Question 10

State what screening programmes do

Answer 10

• Search for specific disease alleles within genomes

Question 11

State where sequences genes are stored

Answer 11

• Gene banks

Question 12

Define bioinformatics

Answer 12

• the digital storage of large quantities of biological data

Question 13

State four features of bioinformatics

Answer 13

• Sequences for existing healthy or disease alleles are stored
• Allows for rapid comparison between newly discovered sequences and stored ones
• Existing protien primary structure stored
• Can model possible 3D shapes of new proteins coded for the the newly discovered alleles

Question 14

Define tandem repeats

Answer 14

• Repetitive segments of DNA that do not code for proteins

Question 15

Define DNA profiling

Answer 15

• Forensic technique that analyses the non-coding tandem repeat DNA to compare DNA samples

Question 16

Describe the general procedure of DNA profiling for forensics

Answer 16

1) DNA is obtained from the crimescene (through sperm/saliva/blood/bone)
2) Restriction enzymes then digest the DNA which cut the DNA at specific recognition sites forming fragements
3) These fragements are separated by gel electrophoresis and are stained
4) A banding pattern can be seen
5) The suspects DNA is treated with the same resitriction enzyme and subjected to electrophoresis
6) The banding patterns of both DNA are compared to see if theres a match

Question 17

Define banding patterns on DNA

Answer 17

• They are patterns of light and dark transverse bands on a chromosome

Question 18

State the three uses of DNA profiling

Answer 18

1) Forensic science
2) Reveal maternity/paternity
3) Analysis of huntingtons disease/diseases

Question 19

Define polymerase chain reaction (PCR)

Answer 19

• Where a short length of DNA is rapidly and synthetically amplified into millions of copies
• (Artificial DNA replication)

Question 20

State what four factors polymerase chain reaction (PCR) relies on about DNA molecules

Answer 20

1) DNA is made of two antiparrallel backbone strands
2) Each strand has a 5’ end and a 3’ end
3) DNA grows from the 3’ end only
4) Base pairs have complementary base pairing

Question 21

State three ways in which polymerase chain reaction differs from DNA replication

Answer 21

1) Only short sequences of DNA can be replicated, not entire chromosomes
2) It requires primer molecules to make the process start
3) It requires cycles of heating and cooling for : strand separation, binding primer to the strands and for the DNA to be replicated

Question 22

Explain why a primer is needed for polymerase chain rection (PCR) but not for DNA replication

Answer 22

• PCR consists of using single stranded DNA
• DNA polymerase is not able to bind to single stranded DNA on its own, thus PCR requires a primer

Question 23

Describe in detail the steps of polymerase chain reaction (PCR)

Answer 23

1) A sample of DNA is mixed with DNA nucleotides, primers, Mg²⁺ and Taq DNA polymerase
2) The mixture is heated to 95°C to break hydrogen bonds between complementary nucleotide base pairs and thus denature the double stranded DNA into two single strands
3) The mixture is cooled to 68°C so the primer can bind by hydrogen bonding to one end on each single strand of DNA
4) This results in a small section of double stranded DNA at the end of each single strand
5) Taq DNA polymerase can now bind to this double stranded region
6) The temperature is raised to 72°C as its optimum temp for taq DNA polymerase enzyme and to keep DNA single-stranded
7) The tag DNA polymerase then catalyses the addition of complementary DNA nucleotides to the single stranded molecule, starting at the primer end and proceeding in the 5’ to 3’ direction
8) Once tag DNA polymerase has reached the other end of the DNA molecule, a new double stranded DNA forms

Question 24

Describe three uses of polymerase chain reaction (PCR)

Answer 24

1) Tissue typing (the donor and patient tissues can be checked before transplant to reduce the risk of rejection)
2) Detection of mutations (sample of DNA is sampled for presence of a mutation that can lead to a genetic disease)
3) Forsenic science (to identify criminals or parentage)

Question 25

Define electrophoresis

Answer 25

• Process that separates DNA fragements (nucleic acid fragments) or protein molecules of different sizes

Question 26

Describe the process of electrophoresis

Answer 26

1) DNA sample first digested with restriction enzymes to cut them into fragments at specific regions
2) DNA sample placed in a well at the negative electrode end
2) Current is applied for a fixed time
3) DNA fragements migrate from the negative end to the positive end
4) Larger fragments move more slowly than smaller fragments, so smaller fragemnts travel further
5) Radioactive or fluorescent probes are added
6) (If radioactive - nylon blotting/autoradiography used to locate fragments) (if fluorescent - uv light used to locate fragments)

Question 27

Why does DNA have an overall negative charge

Answer 27

• Due to its many negative phosphate groups

Question 28

State how proteins are separated via electrophoresis

Answer 28

• Using the charged detergent (SDS) in electrophoresis

Question 29

Explain the effect of SDS in protein electrophoresis

Answer 29

• SDS added equalises the surface charge on the molecules
• This allows proteins to separate as they move through the gel, according to their molecular mass

Question 30

State the condition that can be tested for by SDS electrophoresis

Answer 30

• Sickle cell anaemia

Question 31

State three uses of probes

Answer 31

1) To locate a specific gene needed for use in genetic engineering
2) To identify the same gene in a variety of different genomes from different species when genome comparing
3) To identify presence or absence of a specific allele for a particular genetic disease

Question 32

Define microarray

Answer 32

• A fixed surface with many different probes
• DNA is applied onto it to reveal mutated alleles that are complementary to the fixed probes

Question 33

State the gel used in electrophoresis

Answer 33

• Agarose gel

Question 34

Define DNA ligase

Answer 34

• The enzyme that catalyses the joining of sugar and phosphate groups within DNA

Question 35

State the steps of **genetic engineering

Answer 35

1) The required gene is obtained